Catechol compounds as dual-targeting agents for fish protection against Ichthyophthirius multifiliis infections.

Aquaculture is one of the fastest growing sectors in global food production, recognized as a significant contributor to poverty alleviation, food security, and income generation. However, the frequent occurrence of diseases caused by pathogen infections result in reduced yields and economic losses, posing a substantial constraint to the sustainable development of aquaculture. Here, our study identified that four catechol compounds, quercetin, luteolin, caffeic acid, and chlorogenic acid, exhibited potent antiparasitic effects against Ichthyophthirius multifiliis in both, in vitro and in vivo. The parasite is recognized as one of the most pathogenic to fish worldwide. Using a combination of in silico methods, the dipeptidyl peptidase (DPP) was identified as a critical target for catechol compounds. The two hydroxyl radicals of the catechol group were essential for its binding to and interacting with the DPP protein. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that catechol compounds disrupt pathways associated with the metabolism and growth of I. multifiliis, thereby exerting antiparasitic effects. Furthermore, these compounds attenuated the expression of proinflammatory cytokines in vivo in fish and promoted macrophage polarization toward M2 phenotype by inhibiting the STAT1 signaling pathway. The dual activity of catechol compounds, acting as both direct antiparasitic and anti-inflammatory agents in fish, offers a promising therapeutic approach for combating I. multifiliis infections in aquaculture.

Protection of Grouper Against Cryptocaryon irritans by Immunization With Tetrahymena thermophila and Protective Cross-Reactive Antigen Identification.

Vaccination is an effective method to prevent Cryptocaryon irritans infection. Although some vaccines have been developed, large-scale production of these vaccines is costly. Development of a heterogenous vaccine generated by low-cost antigens is an alternative method. In the present study, grouper immunized with Tetrahymena thermophila, a free-living ciliate that easily grows in inexpensive culture media at high density, showed protective immunity against C. irritans infection. Higher immobilization against C. irritans theronts was detected in T. thermophila-immunized grouper serum, which suggested the existence of a cross-reactive antibody in the serum. By immunoprecipitation and mass spectrometry analyses, tubulin was identified as a potential cross-reactive antigen between C. irritans and T. thermophila. Recombinant T. thermophila tubulin protein (rTt-tubulin) and its antibody were prepared, and immunofluorescence showed that both C. irritans and T. thermophila cilia were stained by the anti-rTt-tubulin antibody. Grouper immunized with rTt-tubulin showed a reduced infective rate after the C. irritans challenge. An enhanced level of C. irritans-binding immunoglobulin M (IgM) antibody was detected in serum from rTt-tubulin-immunized grouper. Moreover, specific antibodies were also found in the mucus and tissue culture medium from rTt-tubulin-immunized grouper. Overall, these findings suggested that vaccination with T. thermophila elicits cross-reactive protective immunity in grouper against C. irritans, and T. thermophila may be a potential heterologous antigen for vaccine development.

Potential Efficacy of Chitosan-Poly (Lactide-Co-Glycolide)-Encapsulated Trivalent Immersion Vaccine in Olive Flounder (Paralichthys olivaceus) Against Viral Hemorrhagic Septicemia Virus, Streptococcus parauberis Serotype I, and Miamiensis avidus (Scuticociliate).

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

Dose effects of a DNA vaccine encoding immobilization antigen on immune response of channel catfish against Ichthyophthirius multifiliis.

Channel catfish (Ictalurus punctatus) vaccinated with pcDNA3.1-IAg52b plasmid DNA vaccine encoding immobilization antigen genes of Ichthyophthirius multifiliis (Ich) produced anti-Ich antibodies and were partially protected (20% survival) in a previous study. Here we evaluated whether a higher dose or two doses of pcDNA3.1-IAg52b vaccine could provide better protection for catfish against Ich. Fish were distributed into 6 groups and vaccinated using following schemes: 1.10 µg pcDNA3.1-IAg52b fish-1, 2.20 µg pcDNA3.1-IAg52b fish-1, 3. two doses of 10 µg pcDNA3.1-IAg52b fish-1 with 7 days between doses, 4.20 µg pcDNA3.1 fish-1 (mock-vaccinated control), 5.15,000 live theronts fish-1 (positive control), and 6. non-vaccinated and non-challenge control. Parasite infection levels, serum anti-Ich antibody levels, fish mortality and immune-related gene expression were determined during the trial. Fish vaccinated with a single dose of 20 µg pcDNA3.1-IAg52b fish-1 or two doses of 10 µg fish-1 had higher anti-Ich antibody levels than fish receiving a single dose of 10 µg fish-1. Survival was significantly higher in fish receiving 20 µg vaccine fish-1 (35.6%) or 2 doses of 10 µg fish-1 (48.9%) than fish injected with a single dose of 10 µg fish-1 (15.6%) or mock-vaccinated control (0%). Fish vaccinated at the dose 20 µg fish-1 had higher expression of vaccine DNA in muscle than fish vaccinated with 10 µg fish-1. Fish vaccinated with the DNA vaccine showed higher up-regulation than mock-vaccinated control in the expression of IgM, CD4, MHC I and TcR-α genes during most of time points after vaccination. Further studies are needed to improve efficacy of DNA vaccines by using multiple antigens in the DNA vaccines.

Immune response of channel catfish (Ictalurus punctatus) against Ichthyophthirius multifiliis post vaccination using DNA vaccines encoding immobilization antigens.

The channel catfish (Ictalurus punctatus) immune response against Ichthyophthirius multifiliis (Ich) after vaccination using plasmid DNA vaccines pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, encoding Ich immobilization antigen genes was studied. Parasite infection level, serum anti-Ich antibodies level, fish mortality after theront challenge, and immune-related gene expression were measured. After in vitro transfection of walking catfish gill cells (G1b) with both pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, antigens IAG52A and IAG52B were detected. During the vaccination trial, 76-fold increase in the Iag52b gene expression was observed in the vaccinated fish group h4 post vaccination. Administration of DNA vaccines by IM injection induced significant gene up-regulation in the head kidney, including immunoglobulin M (IgM), cluster of differentiation 4 (CD4), major histocompatibility I (MHC I), and T cell receptor α (TcR-α) from h4 to d5 post immunization. Fish vaccinated with DNA vaccines or theronts showed increased gene expression of the cytokine interferon (IFN-γ), complement component 3 (C3), and toll-like receptor-1 (TLR-1). Anti-Ich antibodies were detected in fish received pcDNA3.1-IAg52a, pcDNA3.1-IAg52b and the combination of both vaccines d10 post vaccination. Fish vaccinated with pcDNA3.1-IAg52b showed mild parasite infection level, partial survival (20%) and longer mean day to death (MDD) after theront challenge. By contrast, a heavy parasite load, 0% survival and short MDD were observed in the sham vaccinated control fish that received pcDNA3.1 (plasmid without genes encoding Ich immobilization antigen). Further research is needed to improve DNA vaccines for Ich that can induce strong protective immunity in fish. Suggested studies include improved transfection efficiency, use of appropriate adjuvants and including additional parasite antigen genes in the plasmid.

Experimental evidence on prevention of infection by the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon using ultraviolet disinfection of rearing water.

In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery-reared juveniles. This study examined the susceptibility of the two parasites to wide-ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small-scale rearing system over a 28-day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 105  µW s/cm2 for I. salmonis and ≥8.40 × 105  µW s/cm2 for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 106  µW s/cm2 succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery-reared juvenile chum salmon.

Effects of clioquinol on the scuticociliatosis-causing protozoan Miamiensis avidus in olive flounder Paralichthys olivaceus.

Scuticociliatosis is a devastating and intractable protozoal disease in olive flounder, leading to a significant loss throughout the year. This study aimed to investigate a systemically effective antiscuticociliatosis agent for olive flounder for better absorption into the infected internal organs. The in vitro and in vivo antiscuticociliatosis effects of clioquinol (CQ) were examined after screening 30 biocidal agents against the highly pathogenic scuticociliate Miamiensis avidus. CQ was the most potent in vitro drug of those tested against cultured M. avidus. CQ was the least toxic in healthy olive flounder among the drugs that exhibit high potencies. In olive flounder, a single intramuscular injection of 40 mg/kg CQ significantly reduced mortality caused by artificial infection with M. avidus, and 10-20 mg/kg CQ increased fish survival times. CQ was also effective in naturally infected scuticociliatosis. Ciliate cell numbers were lower when CQ was injected in most organs, including the brain. CQ was well absorbed by the internal organs after intramuscular injection. This study suggests that CQ can be considered as a potential antiscuticociliatosis agent for systemic administration in olive flounder.

The effect of dietary immunostimulants on the susceptibility of common carp (Cyprinus carpio) to the white spot parasite, Ichthyophthirius multifiliis.

One of the main obstacles in freshwater aquaculture is the parasitic ciliate Ichthyophthirius multifiliis (Ich), the causative agent of white spot disease. The use of immunostimulants as feed additives may be a promising approach to control Ich infection. In the present study, we tested the prophylactic effect of orally administered ß-1,3/1,6-glucan and propolis extract E50 against Ich infection in common carp. In total, 122 fish were separated into three experimental groups fed with a control, 3% ß-glucan and 1% propolis diet for 40 consecutive days, respectively. On day 40, 16 fish per group were individually exposed to Ich theronts and the number of trophonts was counted 5 days post exposure. Relative gene expression of interleukin 1-ß (IL-1-ß) in common carp liver was examined by qPCR. Compared to control, the mean infection intensity was lower in the ß-glucan- and propolis-fed groups; however, the difference was not statistically significant. The relative expression of IL-1-ß significantly decreased in the propolis-fed group at day 10. In the ß-glucan-fed group, a significant IL-1-ß decrease was detected at day 15 compared to control. Although the Ich infection intensity was slightly decreased in both treated groups, and IL-1-ß was moderately down-regulated in the liver of common carp, our results suggest that the applied feeding regime is insufficient to prevent Ich outbreaks in common carp.

Antiprotozoal effects of metal nanoparticles against Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a widespread, ciliated protozoan ectoparasite of fish. In the present study, we investigated the effects of metal nanoparticles on the reproduction and infectivity of free-living stages of I. multifiliis. We determined that ~50% of theronts could be killed within 30 min of exposure to either 20 ng mL-1 gold, 10 ng mL-1 silver or 5 ng mL-1 zinc oxide nanoparticles. Silver and zinc oxide nanoparticles at concentration of 10 and 5 ng mL-1 killed 100 and 97% of theronts, respectively and inhibited reproduction of tomonts after 2 h exposure. Gold nanoparticles at 20 ng mL killed 80 and 78% of tomonts and theronts 2 h post exposure, respectively. In vivo exposure studies using rainbow trout (Oncoryhnchus mykiss) demonstrated that theronts, which survived zinc oxide nanoparticles exposure, showed reduced infectivity compared with control theronts. No mortalities were recorded in the fish groups cohabited with theronts exposed to either nanoparticles compared with 100% mortality in the control group. On the basis of the results obtained from this study, metal nanoparticles particularly silver nanoparticles hold the best promise for the development of effective antiprotozoal agents useful in the management of ichthyophthiriosis in aquaculture.

Rainbow trout (Oncorhynchus mykiss) immune response towards a recombinant vaccine targeting the parasitic ciliate Ichthyophthirius multifiliis.

The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n-terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I-antigen in future vaccine trials.

Live recombinant Lactococcus lactis vaccine expressing immobilization antigen (i-Ag) for protection against Ichthyophthirius multifiliis in goldfish.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, the present study was conducted to evaluate a live recombinant Lactococcus lactis vaccine expressing immobilization antigen (IAG-52X) in protection against I. multifiliis. A 1266 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of I. multifiliis was assembled from six synthetic ohgonucleotides and cloned into pSIP409 and electrotransformed into Lactobacillus plantarum NC8. The recombinant vaccine candidate was then orally fed into goldfish. The expression of immune-related genes: complement component 3 (C3), MHC I, IgM gene in blood from goldfish at different time points after immunization were evaluated. Immunized fish were than challenged with a lethal dose of infectious I. multifiliis. The cumulative mortality and relative percentage survival (RPS) were also determined. Our results showed that the antibody level in the blood and skin of the immunized fish was statistically significant (P < 0.05) in relation to the control groups. Goldfish orally immunized with NC8-pSIP409- IAG-52X had high serum antibody titers that ranged from 32 to 256 after 28d post immunization, while fish fed with NC8-pSIP409 or PBS had no detectable immobilizing antibody response. Expression of IgM, C3, MHC I genes in the group immunized with IAG-52X were significantly (P < 0.05) up regulated as compared with control group, indicating that different immune cells were actively involved in cellular immune response. The results showed that the average survival rate of fish orally immunized with 108 and 106NC8-pSIP409-IAG-52X was 60% and 50% respectively. Therefore, NC8-pSIP409-IAG-52X could become a promising oral vaccine candidate against I. multifiliis.

Molecular immune response of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. The fish surviving natural infections or immunized with live theronts develop strong specific and non-specific immune responses. Little is known about how these immune genes are induced or how they interact and lead to specific immunity against Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after immunization with live theronts of I. multifiliis. The immunized fish showed significantly higher anti-Ich antibody expressed as immobilization titer and ELISA titer than those of control fish. The vast majority of immunized fish (95%) survived theront challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) showed up-regulation from h4 to d6 post immunization, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1ßa, IL-1ßb, IFN-γ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against infection by I. multifiliis.

Essential oils to control ichthyophthiriasis in pacu, Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia.

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 µL L(-1) , 114 µL L (-1) , 227 µL L(-1) and 455 µL L (-1) , which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day(-1) for 5 days using the M. alternifolia EO (50 µL L (-1) ). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 µL L (-1) ) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.

Cynatratoside-C efficacy against theronts of Ichthyophthirius multifiliis, and toxicity tests on grass carp and mammal blood cells.

Infection by Ichthyophthirius multifiliis, a ciliated protozoan parasite, results in high fish mortality and causes severe economic losses in aquaculture. To find new, efficient anti-I. multifiliis agents, cynatratoside-C was isolated from Cynanchum atratum by bioassay-guided fractionation in a previous study. The present study investigated the anti-theront activity, determined the toxicity of cynatratoside-C to grass carp Ctenopharyngodon idellus and mammalian blood cells, and evaluated the protection of cynatratoside-C against I. multifiliis theront infection in grass carp. Results showed that all theronts were killed by 0.25 mg l-1 of cynatratoside-C in 186.7 ± 5.8 min. Cynatratoside-C at 0.25 mg l-1 was effective in treating infected grass carp and protecting naive fish from I. multifiliis infestation. The 96 h median lethal concentration (LC50) of cynatratoside-C to grass carp and 4 h median effective concentration (EC50) of cynatratoside-C to theront were 46.8 and 0.088 mg l-1, respectively. In addition, the hemolysis assay demonstrated that cynatratoside-C had no cytotoxicity to rabbit red blood cells. Therefore, cynatratoside-C could be a safe and effective potential parasiticide for controlling I. multifiliis.

Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.

Teflubenzuron as a tool for control of trichodinids in freshwater fish: Acute toxicity and in vivo efficacy.

In this study we describe the anti-Trichodina effects of teflubenzuron (TFB) for Oreochromis niloticus and for Piaractus mesopotamicus. We also evaluated the acute toxicity, for both species, by using TFB in the concentrations of 700.0, 800.0, 900.0 and 1000.0 mg L(-1) and a control, without the drug. To assess the efficacy of TFB against Trichodina spp., we used the concentrations of 30.0 or 50.0 mg L(-1) for one hour exposure in tilapia, and the concentration of 30.0, 50.0 and 80.0 mg L(-1) for one hour and 50 mg L(-1) for two hours exposures in pacu. Teflubenzuron did not present significant toxicity in either species, with LC50;48h > 1000.0 mg L(-1). The drug effectiveness was observed against four identified Trichodina species: T. magna, T. heterodentata, T. compacta and T. centrostrigeata, with 87.9% parasite reduction with one hour exposure to 50.0 mg L(-1) TFB on O. niloticus and 96.1% with two hours exposure to 50.0 mg L(-1) TFB on P. mesopotamicus. Teflubenzuron is a drug with potential to be used in Brazilian aquaculture; it attends to important requirements, such as low toxicity and high efficacy in controlling Trichodina spp. infection in O. niloticus and P. mesopotamicus.

Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 µL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 µL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress.

Preventive and therapeutic effects of auxotrophic Edwardsiella tarda mutant harboring CpG 1668 motif-enriched plasmids against scuticociliatosis in olive flounder (Paralichthys olivaceus).

Previously generated two auxotrophic genes knockout Edwardsiella tarda (Δalr Δasd E. tarda) was used as a delivery vehicle for CpG 1668 motif-enriched plasmids (pL-CpG30), and potential of the Δalr Δasd E. tarda-mediated delivery of CpG motifs in both prevention and therapeutic treatment of scuticociliatosis caused by Miamiensis avidus in olive flounder (Paralichthys olivaceus) was investigated. The auxotrophic E. tarda mutant with pL-CpG30 plasmids elicited evidently higher survival rates and delayed both onset of mortality and time-to-death of olive flounder against M. avidus challenge. Furthermore, administration of E. tarda mutant that contains plasmids enriched in CpG 1668 motif elicited clearly higher survival rates of fish that were already infected with M. avidus. These results indicate that CpG 1668 plasmids-harboring E. tarda mutant may provide not only preventive measure but also therapeutic approach against scuticociliatosis in olive flounder.

Immunity to Ichthyophthirius infections in fish: a synopsis.

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects freshwater fish. It has been the subject of both applied and basic research for over 100years, which can be attributed to its world-wide distribution and its significant economic impact on both food and aquarium fish production. I. multifiliis serves as a model for studies in fish on innate and acquired immunity, as well as on mucosal immunity. Although an obligate parasite, I. multifiliis is relatively easily passaged from infected to naïve fish in laboratory aquaria, and is easily observed and manipulated under laboratory conditions. It parasitizes the epithelia of the skin and gills, which facilitates in vivo experimentation and quantification of challenge. This review provides a description of both mucosal and systemic innate and adaptive immune responses to parasite infection, a synopsis of host-parasite immunobiology, vaccine research, and suggested areas for future research to address critical remaining questions. Studies in carp and rainbow trout have shown that extensive tissue damage occurs when the parasite invades the epithelia of the skin and gills and substantial focal and systemic inflammatory responses are elicited by the innate immune response. The adaptive immune response is initiated when phagocytic cells are activated by antigens released by the parasite. It is not known whether activated T and B cells proliferate locally in the skin and gills following infection or migrate to these sites from the spleen or anterior kidney. I. multifiliis infection elicits both mucosal and systemic antibody production. Fish that survive I. multifiliis infection acquire protective immunity. Memory B cells provide long-term humoral memory. This suggests that protective vaccines are theoretically possible, and substantial efforts have been made toward developing vaccines in various fish species. Exposure of fish to controlled surface infections or by intracoelomic injection of live theronts provides protection. Vaccination with purified immobilization antigens, which are GPI-anchored membrane proteins, also provides protection under laboratory conditions and immobilization antigens are currently the most promising candidates for subunit vaccines against I. multifiliis.

Ichthyophthiriasis: emphases on the epizootiology.

Ichthyophthiriasis (white spot disease) is an economically important protozoosis caused by Ichthyophthirius multifiliis in freshwater fish. Medication prevention and curation are the main methods to control this disease with vaccines in laboratory, but the efficacy of drugs practically acts on the free-living (nonparasitic) stage of I. multifiliis and can be easily impaired by a variety of environmental factors. Thus, study on the biological properties of I. multifiliis and the complicated ecological relationships between I. multifiliis and other biotic or abiotic factors that influence epidemicity of ichthyophthiriasis will contribute to integrated control of ichthyophthiriasis. In this article, some I. multifiliis biological properties, such as systematic position and life cycle of I. multifiliis, are briefly reviewed, and the seemingly abnormal phenomenon associated with in vitro cultures is specially discussed; then, the epizootiology of ichthyophthiriasis is emphasized, which involves various biotic or abiotic factors that impact the life and action of I. multifiliis. The susceptibility and stress reaction of fish to I. multifiliis infection are stated. Also, the pathogenicity and diagnosis of ichthyophthiriasis were covered, and an overall assessment is finally made on ichthyophthiriasis control.