Immunosuppression in juvenile carp, Cyprinus carpio L.: the effects of the corticosteroids triamcinolone acetonide and hydrocortisone 21-hemisuccinate (cortisol) on acquired immunity and the humoral antibody response to Ichthyophthirius multifiliis Fouquet

Abstract. Juvenile carp, Cyprinus carpio L., were immunized against Ichthyophthirius multifiliis following controlled exposure to the infective theront stage. On challenge, with a potentially lethal dose of theronts, all immunized fish survived, in contrast to 100% mortality in non-immunized fish. Fourteen days following exposure to the challenge infection, immune and non-immune fish were injected intraperitoneally with either triamcinolone acetonide or hydrocortisone 21-hemisuccinate (cortisol) at doses of 100 μg or 10 μg/g body weight. Controls, including immunized and non-immunized fish, were injected with saline or left untreated. Six days following corticosteroid administration, fish were exposed to challenge infections of I multifiliis. All fish treated with corticosteroid developed heavy infections with up to 100% mortality. Repeat experiments gave comparable results. Serum antibody levels recorded in similarly treated fish at intervals of 12 h and/or 1 week following corticosteroid administration were found to be relatively unaffected. The results are discussed in relation to the immune response to I multifiliis .     

Cell-mediated immune response of goldfish, Carassiusaurafus (L.), to Ichthyophthirius multifiliis

Goldfish, Carassius auratus (L.), were intraperitoneally injected with two doses of Ichthyophthirius multifiliis (each containing 2.3 × 10⁶ live tomites) in saline, 30 days apart. The control fish were injected with saline only. Two weeks after the last vaccination, fish of both groups were intradermally injected with 2.5 × 10⁵ live tomites for skin tests. The skin at the antigen-injected site of the vaccinated fish increased in thickness from 8 h onwards and reached a peak at 24 h. Histological study showed a heavy infiltration of mononuclear leucocytes at the antigen-injected site. In contrast, no such changes were encountered in control fish. The migration area of pronephros cells from the vaccinated fish was significantly inhibited, in vitro, compared to controls with the presence of antigens. These findings suggest that the tomite-vaccinated fish produced a cell-mediated immune response. However, vaccinated fish also exhibited significantly higher titres of immobilizing antibodies in their sera and mucus than the controls. Therefore, it is likely that both humoral and cell-mediated immune responses have to work closely together to eliminate the infectious tomites of I. multifiliis which succeed in penetrating the fish epithelial tissues.     

Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus

Tilapias, Oreochromis aureus, were vaccinated twice with live tomites of Ichthyophthirius multifiliis in physiological saline 1 month before spawning (test group). A control group was similarly treated but with physiological saline only. Fertilized eggs were collected from the mouths of both groups and incubated until the young reached the free-swimming stage. Free-swimming fry were also collected immediately after they were expelled from the mother's mouth in other fish of the two groups. The fry were exposed to infective tomites of I. multifiliis and thereafter returned to their holding tanks. All fry obtained from the controls without mouth-brooding died after tomite exposure. However, fry with mouth-brooding before tomite exposure showed 37.3% survival. On the other hand, fry obtained from vaccinated broodstock without mouth-brooding exhibited 78.4% survival which increased to 95.3% with mouth-brooding. The protective immunity is correlated with the titres of anti-I. multifiliis antibodies in the soluble extracts of fry tissues and the mother's plasma. The results clearly indicate that protective immunity against ichthyophthiriasis “ich” in tilapia fry can not only be derived directly from the mother via eggs but also be acquired indirectly from the mouth cavity during the brooding period. The results thus suggest that proper vaccination of mothers before spawning would effectively protect fry against a protozoan disease.     

Attempts to control Ichthyophthirius multifiliis Fouquet (Ciliophora: Ophryoglenidae) by chemotherapy and electrotherapy

Abstract. New methods were studied for the control of Ichthyophthirius muttifiliis Fouquet, one of the most serious parasites of fish. Totals of 272 fish, 354 I. Multifiliis trophozoites and 562,500 tomites were used in the screening of amprolium, ronidizole, and silver nitrate for parasite control. In addition, 1,465 trophozoites and 600 tomites were subjected to pulsed DC electrotherapy trials. Silver nitrate at 0·67 ppm was 100% effective in eradicating tomites, while ronidizole at 750 ppm gave 100% and 97% control of trophozoites and tomites, respectively. Amprolium and electrotherapy were both ineffective against trophozoites and tomites. Silver nitrate has promise as a safe and effective alternative to chemicals currently used to control I. multifiliis .     

Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites

Abstract The antiparasitic effects of piscidin 2, an antimicrobial polypeptide (AMPP) first isolated from mast cells of hybrid striped bass, were tested against three protistan ectoparasites of marine fish (the ciliates Cryptocaryon irritans and Trichodina sp., and the dinoflagellate Amyloodinium ocellatum) and one ciliate ectoparasite of freshwater fish (Ichthyophthirius multifiliis). I. multifiliis was the most susceptible parasite, with all theronts killed at 6.3 microg mL(-1) piscidin 2. The most resistant parasite was Trichodina, where a few cells were killed at 12.5 microg mL(-1), but several were still alive even at 100 microg mL(-1). C. irritans was of intermediate sensitivity, with some theronts killed at 12.5 microg mL(-1) and all killed at 25 microg mL(-1). High parasite density apparently exhausted the piscidin 2 before it could attain its maximal effect, but surviving parasites were often visibly damaged. The lower efficacy of piscidin 2 against marine parasites compared with the freshwater ciliate might be related to the inhibitory effects of high sea water cation levels. The tissue concentration of piscidins estimated in healthy hybrid striped bass gill (40 microg mL(-1)) suggests that piscidin 2 is lethal to the parasites tested at physiological concentrations and is thus an important component of innate defence in fish expressing this type of AMPP.     

An experimental study into the possible protection of fry from Ichthyophthirius multifiliis (Fouquet) infections during mouthbrooding of Oreochromis mossambicus (Peters)

Abstract. The tilapine fish species belonging to the genus Oreochromis brood developing eggs and early fry in their buccal cavities. Though it is obvious that mouth brooding facilitates a physical protection from environmental hazards by keeping fry in this confined environment, it is still not clear whether mouth brooding provides any other type of protection to the early fry against pathogens. Investigations were made to study the possibility of protection of early fry of O. mossambicus against infections of the ciliate parasite Ichthyophthirius multifiliis during mouth brooding. Adult females were effectively immunized against I. multifiliis. Brooding immune females were then challenged with controlled infections of I. multifiliis tomites and the survival of the fry compared to those of challenged non-immune brooding females. The study demonstrated that there was some protection from the infection conferred to fry during mouthbrooding.     

The immune response of Atlantic salmon, Salmo salar L., to the causative agent of bacterial kidney disease, Renibacterium salmoninarum

Abstract. Both under-yearling and post-yearling Atlantic salmon parr produced high agglutinating antibody titres in response to a single intraperitoneal injection of killed bacterial kidney disease (BKD) cells emulsified in. Freund's complete adjuvant (FCA), Low or no response was observed in animals injected with BKD cells in saline or in animals vaccinated by hyperosmotic immersion. Immunological duration was insufficient in fish vaccinated as under-yearling parr to provide protective immunity 2 years later when the fish had become smolts. Atlantic salmon post-yearling parr injected with BKD cells in FCA demonstrated a reduced prevalence of BKD lesions compared to control animals when both were observed as smolts 1 year after vaccination.     

Experimental Cryptobia salmositica (Kinetoplastida) infections in Atlantic salmon, Salmo salar L.: cell-mediated and humoral immune responses against the pathogenic and vaccine strains of the parasite

Hatchery-reared Atlantic salmon, Salmo salar L., were vaccinated intraperitoneally (i.p.) with a live attenuated Cryptobia salmositica vaccine (either 100 000 or 5000 parasites fish⁻¹) and 4 weeks later were challenged with the parasite (either 100 000 or 5000 parasites fish⁻¹). Unvaccinated, infected salmon had high parasitaemias and were anaemic. Fish given a high dose (100 000 parasites fish⁻¹) had higher parasitaemias than fish given the lower dose. Vaccinated fish had low parasitaemias and a mild anaemia, but recovered quickly after challenge. Complement-fixing antibody increased in vaccinated fish after challenge and was highest at 2 weeks post-challenge. The cell-mediated response (both T cells and B cells) was depressed in infected fish until 4 weeks after infection. In vaccinated fish, the humoral response (i.e. B-lymphocytes) was greater than the cell-mediated response (i.e. T-lymphocytes). In contrast, infected fish had a greater cell-mediated than humoral immune response.     

人工感染的刺激隐核虫各期虫体的超微结构

从天然感染的卵圆鲳鲹（Trachiruotus blochii）分离到1株刺激隐核虫（Cryptocaryon irritans）,再经人工感染的方法收集各期虫体,制成电镜样品,对虫体进行超微结构研究。刺激隐核虫质膜的基本结构与淡水的多子小瓜虫的质膜相似,分为3层;外限制膜、外胞膜和内胞膜。刺激隐核虫的质膜小泡内充满大量电子致密物质（EDM）。幼虫、幼体和滋养体均具有复杂的口器,由胞咽、口肋、双动基体口纤毛和线带等组成,但不具备膜口类纤毛虫所具有的独特的细胞器：Lieberkuhn氏器。单动基体的体纤毛存在于幼虫、幼体和滋养体,但在包囊期,体纤毛和口器消失。各期虫体的胞质中具有线粒体、高尔基体和脂肪体等细胞器,有的阶段具有粘液囊、伸缩泡、食物泡、共生细菌等。粘液囊在大小、形态和分布上与多子小瓜虫的不同。文中分析了刺激隐核虫在形态上与多子小瓜虫的诸多差异,认为刺激隐核虫的分类更适合归于原口类（Promotome）,而不是膜口类（Ophryoglenina）。     

Cryptocaryon irritans Brown, 1951 (Ichthyophthiriidae): the ultrastructure of the somatic cortex throughout the life cycle

Abstract. Cryptocaryon irritans was isolated from Grammistes sexlineatus and maintained in aquaria under controlled conditions using the mullet, Chelon labrosus (Risso), as an experimental host. The pellicle and cortical cytoplasm of the trophont, tomont, tomite and theront stages were investigated with the aid of transmission electron microscopy. The general structural organization of the pellicle resembles that of the freshwater holotrich, Ichthyophthirius multifiliis ; however, the pellicular alveoli are characterized by the accumulation of electron-dense material in the trophont, tomite and theront, this material being lost in the newly-encysted tomont. The outer membranes of the pellicle are infolded into the alveoli of the trophont. Cilia are present at all stages of the life cycle, although at encystment their numbers are greatly reduced by shedding. Mitochondria, mucocysts and Golgi-like cisternae are distributed throughout the cortical cytoplasm. Mucocysts differ from those of I. multifiliis in size, shape and distribution and are of uniform electron density. The role of the pellicle and cortical cytoplasm is discussed in relation to the invasion of the fish epidermis, possible intertransferance of materials between parasite and host and the process of encystment.     

Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyophthirius multifiliis

Abstract Channel catfish, Ictalurus punctatus (Rafinesque), were immunized with Ichthyophthirius multifiliis (Ich) theronts and trophonts, and the immune response and host protection against both homologous and heterologous serotypes of Ich were evaluated. Immunizations were done with two immobilization serotypes (ARS4 and ARS6) of live theronts by bath immersion (trial I) and with sonicated trophonts by intraperitoneal (i.p.) injection (trial II). Cutaneous and serum antibody titres against Ich following immunization were measured and survival of catfish was determined after theront challenge. Theronts were immobilized by the antiserum from fish immunized with homologous theronts or trophonts, but not by the serum of fish immunized with the heterologous serotype. Serum from fish immunized by immersion with live theronts showed higher enzyme-linked immunosorbent assay titres against both homologous and heterologous serotypes than fish immunized by i.p. injection of trophonts. Channel catfish immunized by immersion with live theronts or by i.p. injection with sonicated trophonts developed an immune response against Ich and provided cross-protection against challenge from both serotypes (ARS4 and ARS6) of the parasite. Sonicated trophont antigens in aqueous solution by i.p. injection could stimulate an immune response in fish, but the immunity was of short duration.     

Ichthyophthiriasis in carp, Cyprinus carpio L.: fate of parasites in immunized fish

Abstract. The host-parasite relationship between O-group carp and the ciliatc Ichthyophthirius multifiliis Fouquet was investigated, with the specific aim of characterizing the fate of parasites encountering immunized fish. Carp were immunized by repeated controlled infections; immunized fish and control fish naive to I. multifiliis were then infected in the caudal fin epidermis by a single controlled exposure to theronts, which were applied in a droplet suspension to the tail surface. The number of parasites present within the caudal fin was monitored over a subsequent 5-day period by means of in situ parasite mapping. Results indicated that, contrary to previous reports, theronts penetrated the skin of immunized fish in numbers comparable to those of fish receiving a primary infection. However, the majority of parasites which penetrated immune skin did not complete normal development; 79% of the parasites which had initially penetrated the immune skin were not relocated within 2h of exposure, and since no parasite material was detected at penetration sites, it was concluded that these parasites had prematurely exited the skin rather than been killed in situ. Subsequently, these sites became populated by leucocytes, predominantly macrophages, and the infiltrations continued for up to 5 days after the initial exposure. In contrast, at sites where mature trophonts had exited the skin of fish following a primary infection, more diffuse leucocytic infiltrations were recorded, and these were predominated by neutrophils. Differences in the response to parasite exit from immunized and previously unexposed control fish skin are discussed, with particular reference to the mode of protection and the fate of parasites encountering immune fish.     

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.     

Protection against carp erythrodermatitis following bath or subcutaneous exposure to sublethal numbers of virulent Aeromonas salmonicida subsp. nova

Abstract. A bath challenge system was used to infect carp. Cyprinus carpio L., with Aeromonas salmonicida subsp. nova , the causative agent of carp cruthrodermatitis. Bath-challenged fish became infected with the bacterium exihibitinng typical signs of the disease, Carp that were sublethally bath exposed became infected and exhibited some skin lesions, but after one week, these quickly healed and the animals fully recovered from the infection, Naive fish that had not been previously exposed to the bacterium had mortalities of 100% when infected by the subcutaneous route and 40–60% by the bath route of infection. Carp that received sublethal infections were able to withstand subsequent lethal infection and recover regardless of the route of infection. Sublethally bath-exposed carp were protected from subsequently lethal challenges of A. salmonicida subsp. nova for at least 5 months.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Ichthyophthiriasis of Atlantic salmon, Salmo salar L., at the Montta Hatchery in northern Finland in 1978–1979

Abstract. The occurrence of the ciliated protozoan Ichthyophthirius multifiliis on Atlantic salmon yearlings at a hatchery in northern Finland is described. During August 1978 a severe epizootic occurred in two earth ponds resulting in mortality of over 50%. Water temperature at this time was about 15°C. Fish were treated with a 1:4000 formalin bath administered every 3 days. Parasites disappeared from the fish by October after which they were refractory to further infection. The parasite occurred in other stocks in 1979 but was successfully controlled with formalin. Some aspects of the ecology of I, multifiliis are discussed.     

Efficacy of Vibrio anguillarum antigen administered by intraperitoneal injection route in Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), were immunized by the intraperitoneal (i. p.) injection route with formalin‐killed whole cells of Vibrio anguillarum that originated from a diseased fish. Fifty days later, a booster vaccination was given by the same route. Control fish were similarly treated with sterile phosphate‐buffered saline. The efficacy of vaccination was evaluated based on protection against two bacterial challenges and immune responses (both specific and non‐specific). The challenges were performed by i. p. injection with V. anguillarum or V. parahaemolyticus. The results indicated that the vaccinated fish showed higher non‐specific immune activity than the unvaccinated fish. The effects of vaccinations on the phagocytic activity of phagocyte, bactericidal and lysozyme activities were notable, especially on bactericidal activity. Determined by ELISA, antiserum of vaccinated fish displayed high antibody titres. The vaccination conferred protection against V. anguillarum challenge (81.25–93.75% relative percentage survival (RPS)). The RPS was 46.15–53.85% against V. parahaemolyticus challenge, indicating some degree of cross‐protective immunity.     

Protection against atypical furunculosis in Atlantic halibut,Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.     

Vaccination trials of sea bass,Dicentrarchus labrax (L.), against Photobacterium damsela subsp. piscicida,using novel vaccine mixtures

Bacterial cells of the marine fish pathogen Photobacterium damsela subsp. piscicida were grown in novel culture media. A mixture of whole cells and extracellular components was inactivated and used in bath, intraperitoneal (i.p.) and oral vaccination of sea bass, Dicentrarchus labrax, employing two sizes of fish. A commercial vaccine was used for comparative purposes. Control and immunized fish were either bath or intraperitoneally challenged 6 and 12 weeks post-vaccination. Small fish had significantly higher relative percentage survival with the novel vaccine mixture both at 6 and 12 weeks post-vaccination by bath, in comparison with the commercial vaccine. No protection was afforded at 6 or 12 weeks post-immunization by either vaccine after challenge via i.p. injection. Sea bass (1.5-2 g) intraperitoneally vaccinated with various adjuvanted vaccine mixtures were not protected against pasteurellosis. In contrast, larger sea bass (20 g) benefited from vaccination with the novel vaccine mixtures. Intraperitoneal challenge with the pathogen resulted in protection in both fish groups vaccinated with novel vaccine mixtures, whereas control fish suffered high mortalities (> 80%). Orally vaccinated fish were immersion challenged with the pathogen. At 6 and 12 weeks post-vaccination the control fish had a high mortality and the fish vaccinated with the novel vaccine mixture achieved good protection.     

Passive immunization of tilapia, Oreochromis niloticus (L.), with anti-Streptococcus iniae whole sera

Passive immunization of tilapia, Oreochromis niloticus, was conducted to determine whether anti- Streptococcus iniae whole sera (ASI), heat inactivated anti- S . iniae whole sera (HIASI) and normal whole sera (NWS) were protective when intraperitoneally (i.p.) injected into tilapia. The ASI was produced in tilapia actively immunized (challenged) with virulent S. iniae by i.p. injection. An antibody response against S. iniae was demonstrated by enzyme linked immunosorbent assay (ELISA) and 18% of the immunized fish died because of the S. iniae infection. The actively immunized tilapia demonstrated a secondary antibody response and immunity to S. iniae after challenge with S. iniae by i.p. injection. Survival was 100% in the actively immunized fish. The NWS was obtained from tilapia free of ASI antibody and susceptible to S. iniae infection (40% mortality). In two separate experiments, significantly higher mortality was noted in tilapia passively immunized with NWS (33 and 53%) and phosphate buffered saline (PBS) (30 and 60%), in comparison with mortalities of 0 and 10% or 3.3 and 6.7% in the fish passively immunized with ASI or HIASI 14 days after S . iniae infection by i.p. injection ( P  = 0.0003 and 0.0023). Results suggest that immunity provided by ASI and HIASI was because of antibody against S. iniae . Inactivation of complement in the HIASI treatment further suggests that ASI antibody plays a primary role in immunity against S. iniae infection.