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.

Effects of dietary poly-ß-hydroxybutyrate supplementation on the growth, immune response and intestinal microbiota of soiny mullet (Liza haematocheila).

Soiny mullet (Liza haematocheila) is an important economic fish species in China, but stress and diseases have seriously restricted its culture. There are no effective methods including vaccines to prevent or control these diseases. Alternative methods should be employed, such as using novel immunostimulant poly-ß-hydroxybutyrate (PHB). The present study aimed to evaluate effects of dietary PHB supplementation on the growth, antioxidant enzymes activity, immune-related genes expression and intestinal microbiota in soiny mullet. The fish was fed for 30 or 60 days with six diets at different PHB supplementation of 0, 0.5, 1, 2, 4 and 8%, named as groups P0, P0.5, P1, P2, P4 and P8. The results showed that the weight gain and specific growth rate of fish in P2 and P0.5 groups were significantly higher than those in control P0 group at 30 and 60 days, respectively (P < 0.05). The antioxidant enzymes activity of catalase and superoxide dismutase in serum were significantly increased in P0.5/P1/P2 groups after 30 days. The transcriptional levels of penicillin-binding protein A and interleukin-8 analyzed by qRT-PCR were significantly upregulated in P2 and P4 groups compared to those in P0/P0.5/P1/P8 groups at 30 days. The transcriptional level of major histocompatibility complex class II in P2 group was significantly upregulated, and aldehyde oxidase downregulated compared to P0 group. Intestinal microbiota analysis by Illumina high-throughput sequencing showed that the microbiota diversity was not changed significantly, but the microbiota structure shifted significantly post PHB treatment. At the phyla level, Firmicutes and Proteobacteria were predominant in both P0 and P2 groups. At the genus level, the relative abundance of Bacillus spp. in P2 group increased significantly, and abundance of Achromobacter spp. decreased significantly. KEGG pathway analysis by PICRUSt showed that oral administration PHB significantly upregulated abundances of genes responsible for 10 pathways and downregulated genes involved in 17 pathways. In conclusion, soiny mullet fed with 2% PHB supplemental diets for 30 days showed better growth performance, higher antioxidant enzymes activity and immune-related genes expression. Their regulation of growth and immunity might be related with the intestinal microbiota change post PHB supplementation. It will provide very useful basic information to study the regulation mechanism of PHB in aquatic animals, and provide good green method to prevent disease in soiny mullet.

Efficacy and antiparasitic mechanism of 10-gingerol isolated from ginger Zingiber officinale against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis is a ciliate parasite of freshwater fish with a global distribution and results in severe economic losses in aquaculture. The present study aimed to investigate the efficacy and antiparasitic mechanism of active compounds isolated from Zingiber officinale against I. multifiliis. Three compounds were isolated from the Z. officinale extract and identified as 10-gingerol, 6-dehydroshogaol, and 6-dehydro-10-gingerol. 10-gingerol demonstrated the greatest antiparasitic efficacy in vitro. 10-gingerol resulted in 100% mortalities of theronts, nonencysted tomonts, and encysted tomonts at concentrations of 2, 8, and 16 mg/L, respectively. 10-gingerol significantly reduced theronts infectivity (p < 0.05) at a concentration of 1 mg/L, and it was effective in treating infected grass carp and protecting naïve fish from I. multifiliis infestation at a concentration of 4 mg/L. The antiparasitic mechanism might be attributed to the increase of intracellular osmotic pressure, accumulation of free radicals, and membrane damage of I. multifiliis post 10-gingerol treatment. The study demonstrated that 10-gingerol had the potential as a therapeutic agent against I. multifiliis.

Analysis of agglutinants elicited by antiserum of channel catfish immunized with extracellular proteins of virulent Aeromonas hydrophila.

Motile Aeromonas septicemia (MAS), caused by new virulent Aeromonas hydrophila (vAh) strains, has been one of the major diseases in channel catfish in recent years. Previous studies showed that channel catfish developed immunity against vAh infection after immunization with the pathogen's extracellular proteins (ECP). To understand the mechanisms associated with the immunity, anti-ECP fish serum (antiserum) was analyzed in this study. Our results revealed that the antiserum elicited agglutination of both ECP and cells of vAh. Five fish proteins were identified in ECP agglutinants, including two innate immunity associated proteins (serotransferrin and rhamnose-binding lectin), two immunoglobulin M (IgM) molecules (IgM heavy chain and light chain) and a constitutively-produced protein (warm temperature acclimation protein). More than 68 vAh proteins in ECP were recognized and caused to aggregate by IgM in the antiserum. IgM was isolated from vAh cell agglutinants and the native IgM was shown to form a tetramer that was responsible for bacterial agglutination. Immunoblotting analysis indicated that the isolated native IgM was able to recognize some proteins in ECP, such as aerolysin and hemolysin (in the form of a high molecular weight heterologous polymer). Gene expression analysis by quantitative PCR showed that fish immunized with vAh ECP had more transcripts of genes coding for IgM, serotransferrin and rhamnose binding lectin than mock-immunized fish. Both innate and antibody-mediated immune responses in serum and expressed genes contributed to fish immunity upon immunization with ECP. Results of this study shed light on the versatility of vAh antigens and catfish IgM, which would help identify specific antigens for vaccine development and antigen specific antibodies in catfish.

Biofloc technology (BFT): An alternative aquaculture system for prevention of Cyprinid herpesvirus 2 infection in gibel carp (Carassius auratus gibelio).

Gibel carp (Carassius auratus gibelio), a major aquaculture species in China, has emerged in a seriously epizootic disease caused by Cyprinid herpesvirus 2 (CyHV-2). There are no effective methods to prevent or control this serious disease. Biofloc technology (BFT) can improve water quality, reduce pathogens introduction, enhance cultured species immunity and disease resistance. In this study, a 30-day experiment was conducted to investigate the effect of BFT on innate immune response and disease resistance of gibel carp against CyHV-2 infection. Gibel carp was cultured at different total suspended solid (TSS) concentrations of 10, 300, 600, 800 and 1000 mg L-1, which were named as groups BF0, BF300, BF600, BF800 and BF1000. Results showed that fish in groups BF600/800 had significantly higher weight gain (WG) and specific growth rate (SGR) than them in control group (BF0). The transcriptional levels of seven immune-related genes in BF300/600/800 groups, including myeloid-specific- peroxidase (MPO), keratin 8 (KRT 8), dual specificity phosphatase 1 (DUSP 1), interleukin-11 (IL-11), intelectin (ITLN), purine nucleoside phosphorylase 5α (PNP 5α) and c-type lysozyme (c-lys), were up-regulated significantly compared to BF0 group. Furthermore, cumulative mortality of gibel carp in BF600 group after being challenged with CyHV-2 reduced significantly. In vivo viral replication in kidney demonstrated that CyHV-2 load at 168 h post injection in BF600 group was significantly higher than that in BF0 group. In conclusion, BFT could improve growth, immune response and disease resistance of gibel carp, and the effect was related with TSS concentration. The optimal TSS concentration of 600-800 mg L-1 was recommended in the present study.

Grass carp which survive Dactylogyrus ctenopharyngodonid infection also gain partial immunity against Ichthyophthirius multifiliis.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are 2 important ectoparasites of fish. Both parasites can induce an immune response in fish that leads to a decrease in parasitic infection intensity and the development of resistance against parasitic reinfection. The present study evaluated whether grass carp Ctenopharyngodon idella that survived a D. ctenopharyngodonid infection could develop immunity against infection by D. ctenopharyngodonid and I. multifiliis. The results demonstrated that when grass carp were infected with D. ctenopharyngodonid, the number of red blood cells and the percentages of thrombocytes, monocytes, and neutrophils in the white blood cells increased significantly in the early stage of infection. The percentage of lymphocytes increased over time following parasitic infection. The mean infection intensity of D. ctenopharyngodonid decreased to 0 on Day 28. The activities of serum acid phosphatase, alkaline phosphatase, lysozyme, and superoxide dismutase increased significantly after D. ctenopharyngodonid infection. In addition, the grass carp that survived a previous D. ctenopharyngodonid infection could completely resist D. ctenopharyngodonid reinfection and partially resist I. multifiliis infection.

Transcriptome analysis and discovery of genes involved in immune pathways from coelomocytes of Onchidium struma after bacterial challenge.

Onchidium struma widely distributes in subtidal and low-tidal zones, which is considered to be an economical species with rich nutrition, a valuable biomonitor for heavy metal pollution and a representative species for evolution from ocean to land. However, there is limited genetic information available for O. struma development. This study compared transcriptomic profiles of coelomocytes from normal and bacteria infected O. struma by Illumina-based paired-end sequencing to explore the molecular immune mechanism of O. struma against bacterial infection. After assembly, a total of 92,450 unigenes with an average length of 1019 bp were obtained. Approximately 34,964 (37.82%) unigenes were annotated in the Nr NCBI database and 40.1% of unigenes were similar with that of Aplysia californica. Among them, 7609 unigenes were classified into three Gene Ontology (GO) categories: biological process (3250 unigenes, 42.7%), cellular component (2,281, 30.0%) and molecular function (2078 unigenes, 27.3%). A total of 22,776 unigenes were aligned to the Clusters of Orthologous Groups (COG) of proteins and classified into 25 functional categories. Following bacterial infection, 10,623 differently expressed unigenes (DEGs) were identified, including 7644 up-regulated and 2979 down-regulated unigenes. Further KEGG analysis annotated 11,681 DEGs to 42 pathways, and 11 pathways were identified to be related with diseases and immune system. To our knowledge, it was first time to analyze transcriptome profiles of O. struma. Results of the present study will provide valuable theoretical resources for future genetic and genomic research on O. struma. The research results will be helpful for improving the efficiency and quality of artificial breeding, establishing genetic linkage map, and enhancing health management for this species.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

Expression of immune genes in systemic and mucosal immune tissues of channel catfish vaccinated with live theronts of Ichthyophthirius multifiliis.

Ichthyophthiriasis caused by Ichthyophthirius multifiliis (Ich) has a worldwide distribution and affects most freshwater fishes. Fish surviving natural infection and/or immunized with Ich develop strong innate and adaptive immune responses. However, there is a lack of the knowledge regarding immune gene expression patterns in systemic and mucosal immune tissues, and how immune genes interact and lead to innate and adaptive immune protection against Ich infection in fish. The objective of this study was to investigate the expression of innate and adaptive immune-related genes in systemic (liver, spleen) and mucosal (gill, intestine) tissues of channel catfish over time following vaccination with live Ich theronts. The vaccinated fish showed significantly higher antibody titers and survival (95%) than those of mock immunized fish. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 h (h4) to 2 days (d2) post-vaccination in systemic immune tissues. Immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) were more highly upregulated and remained upregulated for longer duration in systemic tissues than in mucosal tissues of the vaccinated fish. The cytokine genes IL-1ßa and IFN-γ were rapidly upregulated in both systemic and mucosal tissues of vaccinated fish, with peak expression from h4 to d1 post-vaccination. Toll-like receptor genes TLR-1 and TLR-9 showed relatively stable upregulation in the gill of immunized fish following vaccination. Results of this study revealed the molecular immune responses in mucosal and systemic tissues of vaccinated fish and demonstrated that Ich vaccination resulted in innate and adaptive immune responses against Ich infection.

Antiparasitic efficacy of curcumin from Curcuma longa against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis is a ciliated parasite that elicits great economic losses in aquaculture. In the present study, a polyphenol compound, curcumin, was obtained from the rhizome of Curcuma longa by bioassay-guided isolation based on the efficacy of anti-I. multifiliis theronts. Anti-I. multifiliis efficacy of curcumin was evaluated in vitro and in vivo. Curcumin resulted in 100% mortality of I. multifiliis theronts at a concentration of 1mg/L within 21.7±1.2min and killed all tomonts at 8mg/L within 31.0±1.0min. Curcumin at 4mg/L for 16h exposure can completely terminate the reproduction of tomonts. The pretreatment with curcumin at concentrations of 0.5, 0.25, and 0.125mg/L for 2h significantly reduced the infectivity of I. multifiliis theronts. Curcumin at 4mg/L completely cured the infected grass carp and protected naive fish from I. multifiliis infection after 10days exposure. The 4h median effective concentration (EC50) of curcumin to I. multifiliis theronts and the 5h EC50 of curcumin to I. multifiliis tomonts were 0.303mg/L and 2.891mg/L, respectively. The 96h median lethal concentration (LC50) of curcumin to grass carp was 56.8mg/L, which was approximately 187.4 times EC50 of curcumin to theronts and 19.6 times EC50 of curcumin to tomonts. The results demonstrated that curcumin has the potential to be a safe and effective therapeutant for controlling ichthyophthiriasis in aquaculture.

Chitin degradation and utilization by virulent Aeromonas hydrophila strain ML10-51K.

Virulent Aeromonas hydrophila (vAh) is one of the most important bacterial pathogens that causes persistent outbreaks of motile Aeromonas septicemia in warm-water fishes. The survivability of this pathogen in aquatic environments is of great concern. The aim of this study was to determine the capability of the vAh strain ML10-51K to degrade and utilize chitin. Genome-wide analysis revealed that ML10-51K encodes a suite of proteins for chitin metabolism. Assays in vitro showed that four chitinases, one chitobiase and one chitin-binding protein were secreted extracellularly and participated in chitin degradation. ML10-51K was shown to be able to use not only N-acetylglucosamine and colloidal chitin but also chitin flakes as sole carbon sources for growth. This study indicates that ML10-51K is a highly chitinolytic bacterium and suggests that the capability of effective chitin utilization could enable the bacterium to attain high densities when abundant chitin is available in aquatic niches.

Draft Genome Sequence of Pseudomonas mosselii Gil3, Isolated from Catfish and Antagonistic against Hypervirulent Aeromonas hydrophila.

Pseudomonas mosselii Gil3 was isolated from a catfish that survived from lethal challenge with hypervirulent Aeromonas hydrophila (vAh). When assayed in vitro, the bacterium showed antagonism against vAh. Sequence analysis revealed that the genome of P. mosselii Gil3 encodes numerous aromatic metabolism pathways and proteins for biosynthesis of antimicrobial compounds.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge.

The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by an emergent, high virulent Aeromonas hydrophila (vAh) in channel catfish, Ictalurus punctatus Adipose fin clipped catfish were challenged with vAh using a waterborne challenge method, and the distribution of vAh over a time course was detected and quantified using real-time polymerase chain reaction. The results showed that 77.8% of fish died within 48 h post challenge with mean day to death of 1.5 days. At 2 h post challenge, vAh (inferred from genomic DNA copies or genome equivalents) was detected in all external and internal tissues sampled. Gill had the highest vAh cells at 1 h post challenge. Spleen harbored the most vAh cells among internal organs at 4 h post challenge. The tissues/organs with most vAh cells detected at 8 h post challenge were adipose fin, blood, intestine, kidney and skin, while liver showed the highest vAh cells at 24 h post challenge. These results suggest that vAh was able to rapidly proliferate and spread, following wound infection, through the fish blood circulation system and cause mortality within 8-24 h.

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.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

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.

Comparison of immune response of Pacific white shrimp, Litopenaeus vannamei, after multiple and single infections with WSSV and Vibrio anguillarum.

Our previous study demonstrated that Pacific white shrimp (Litopenaeus vannamei) infected by multiple pathogens showed higher mortality and death occurred more quickly than those infected by a single pathogen (Jang et al., 2014). For better understanding the defense mechanism against white spot syndrome virus (WSSV) and Vibrio anguillarum, immune responses of shrimp were evaluated in this study. The mRNA expression levels of five immune-related genes were analyzed by quantitative reverse real-time PCR, which included proPO-activating enzyme 1 (PPAE1), PPAE2, proPO activating factor (PPAF), masquerade-like serine proteinase (Mas) and ras-related nuclear gene (Ran). Results demonstrated that the transcription was suppressed more intensively in the multiple infection group than those in single infection groups. The transcriptional suppression was directly related to the higher mortality. The hypoimmunity could benefit pathogen invasion, replication and release of toxin in vivo. Results in this study will help to understand immune defense mechanism after shrimp were infected by multiple pathogens in aquaculture.

Identification and effect of two flavonoids from root bark of Morus alba against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis (Ich) is an important ciliate that parasitizes gills and skin of freshwater fish and causes massive fish mortality. In this study, two flavonoids (kuwanons G and O) with anti-Ich activity were isolated by bioassay-guided fractionation from the root bark of Morus alba, an important plant for sericulture. The chemical structures of kuwanons G and O were elucidated by spectroscopic analyses. Kuwanons G and O caused 100% mortality of I. multifiliis theronts at the concentration of 2 mg/L and possessed a median effective concentration (EC50) of 0.8 ± 0.04 mg/L against the theronts. In addition, kuwanons G and O significantly reduced the infectivity of I. multifiliis theronts at concentrations of 0.125, 0.25, 0.5, and 1 mg/L. The median lethal concentrations (LC50) of kuwanons G and O to grass carp were 38.0 ± 0.82 and 26.9 ± 0.51 mg/L, which were approximately 50 and 35 times the EC50 for killing theronts. The results indicate that kuwanons G and O have the potential to become safe and effective drugs to control ichthyophthiriasis.