Live Theronts Research Articles

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 responses of channel catfish against parasite Ichthyophthirius multifiliis following theront vaccination

Abstract Ichthyophthirius multifiliis (Ich) is a severe fish parasite and results in heavy losses of freshwater fish. The fish surviving natural infections or vaccinated with live theronts develop strong immune responses. Little is known about how immune genes are induced or how they interact and lead to specific immunity against I. 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 vaccination with live theronts of I. multifiliis. The vaccinated fish showed significantly higher anti-Ich antibody than those of control fish. The vast majority of immunized fish (95%) survived the parasite challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hours (h4) to 2 days (d2) post vaccination. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II β, TcR-α, and TcR-β) showed up-regulation from h4 to d6 post vaccination, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1βa, IL-1βb, IFN-□ 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 I. multifiliis infection.

Gökkuşağı alabalıkları (Oncorhynchus mykiss)’nda beyaz benek hastalığına (Ichthyophthirius multifiliis) karşı bağışıklık kazandırılması

In this study, the humoral immune responses and host protection of rainbow trout, O. mykiss (Walbaum, 1792) against Ichthyophthirius multifiliis (Ich) were determined after vaccination with live theronts, sonicated trophonts, adjuvant added heat inactived theronts. Immunizations with live theronts, sonicated trophonts or adjuvant added heat inactive theronts by intraperitoneal (I.P.) injection. Serum immobilization titres were determined 21 days post-immunization. Four weeks after immunization, the number of trophonts were assessed in rainbow trout skin four days after challenge. Mean of serum immobilization titres levels were significantly higher (P<0.05) in fish immunized with live theronts (1/67,4) and sonicated trophonts (1/29,7) by I.P. injection, than in the fish immunized with adjuvant added heat inactivated theronts (1/11,4) and PBS (1/9,42) by I.P. injection. Mean number of the trophonts that can penetrated to skins were significantly lower (P<0.05) in fish administered live theronts (21 trophonts/fish) and sonicated trophonts (29,13 tr/fish) by i.p. injection, than in the fish immunized with adjuvant added heat inactivated theronts (48,53 tr/fish) and PBS (54,07 tr/fish) by I.P. injection. Therefore, partial protection was noted on those fish. There was found positive correlation between higher levels of immobilization titres and partial protection in the immunized fish.

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 β, TcR-α, and TcR-β) 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-1βa 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.

Expression of immune genes in skin of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The objective of this study was to evaluate differential expression of innate and adaptive immune genes, including immunoglobulin, immune cell receptor, cytokine, inflammatory protein, toll-like receptors (TLR) and recombination-activating gene (RAG) in skin from channel catfish, Ictalurus punctatus after immunization with live theronts of Ichthyophthirius multifiliis (Ich) by intraperitoneal injection. The immunized catfish showed significantly higher survival rate (95%) than those of mock-immunized control fish (0% survival) after the theront challenge. The gene expression of innate immune system, such as cytokines (IL-1β type a, IL-1β type b, IFN-γ, TGF1-β and TNF-α) and inflammatory proteins (NF-kB and iNOS 2), showed significant upregulation at day 1 (D1) post-immunization. Expression of TLR genes exhibited a rapid increase from hour 4 (h4) to D10 post-immunization. Genes of the adaptive response, such as the cell receptor MHC I, CD8+ , CD4+ and TCR-α, showed upregulation at D1, D6 and D10. The TCR-β expression increased rapidly at h4 and remained upregulated until D10. Immunoglobulin IgM upregulation was detected from h4 until D2 while IgD expression was increased from D1 until D10. Rapid upregulation of innate and adaptive immune genes in skin of catfish following live theront vaccination was demonstrated in this study ultimately resulting in significant protection against Ich infection.

Histopathological analyses of native silver catfishRhamdia quelen(Quoy and Gaimard, 1824) immunized against and challenged with live theronts ofIchthyophthirius multifiliis

As an alternative to treating with chemicals, immunization using Ichthyophthirius multifiliis as a vaccine has been studied in fishes that were often affected with white spot diseases also to understand the possible changes to the tissue caused by the vaccine. The focus of this study was the analysis of the influence of immunization via intraperitoneal injection (i.p.) and via immersion bath (im.b.) on the histopathology of R. quelen after being challenged with live theronts of I. multifiliis distributed in: control (non-immunized and non-challenged); non-immunized and challenged with 12,000 theronts/fish; non-immunized and challenged with 22,000 theronts/fish; immunized and challenged with 12,000 theronts/fish; immunized and challenged with 22,000 theronts/fish. Water quality was measured in each assay, with 300 fingerlings distributed among 15 tanks with 20 fish in each of three replicates. Six days after challenge, samples for histopathological and parasitological analyses were collected. In both i.p. and im.b. fish the prevalence of I. multifillis in the gills was higher in the non-immunized fish (33.33% and 27.77%, respectively). Melanomacrophages were present in 53% of the samples of i.p. non-immunized fish. Fish im.b. immunized and challenged showed more atrophied areas in the hepatocytes. Higher numbers of melanomacrophages in the i.p. non-immunized fish kidneys were observed compared to control. The results showed no difference in the gill lesions of either immunized or non-immunized fish compared to control. Histological alterations in the organs of silver catfish were considered light, except in the liver that presented significant atrophy and hypertrophy of hepatocytes after immunization via i.p.

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 4hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II β, TcR-α, and TcR-β) 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-1βa, IL-1βb, 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.

Hemato-immunological and biochemical parameters of silver catfish Rhamdia quelen immunized with live theronts of Ichthyophthirius multifiliis.

This study investigated the immunization by intraperitoneal injection (i.p.) (assay I) and immersion bath (assay II) with live theronts of Ichthyophthirius multifillis in Rhamdia quelen and its influence on the hemato-immunological and biochemical parameters. Fish were divided in control (non immunized no challenged); non immunized and challenged with 12,000 theronts/fish; non immunized and challenged with 22,000 theronts/fish; immunized and challenged with 12,000 theronts/fish; immunized and challenged with 22,000 theronts/fish. Six days after challenge, either in the assay I or in the assay II the prevalence of I. multifillis in the gills was higher in non immunized fish (33.33% and 27.77%, respectively). In the assay I showed higher numbers of thrombocytes, leukocytes, lymphocytes, neutrophils and monocytes 20 days after injection and lower numbers after challenge. The immunoglobulin values were higher in fish non immunized. Fish immunized by immersion bath (assay II) showed greater values of catalase (CAT) in the liver (1245.49 U/mgprt) when compared to i.p. (198.79 U/mgprt). The levels of CAT in the liver of fish from the assay II were greater (1738.47 U/mgprt) 14 days after immunization than that observed 21 days after (1114.26 U/mgprt). The vaccination by i.p method showed influence on the hematological parameters. On the other hand, the immersion bath vaccination showed greater influence on the catalase activity in the liver. The results showed that new parameters like total protein, immunoglobulin and antioxidant enzymes could be considered in evaluating the host response to infection.

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.

Minimum effective concentrations of formalin and sodium percarbonate on the free-living stages of an Australian isolate of Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate protozoan, is a common cosmopolitan parasite of freshwater teleosts and is a recurring problem during the summer months on Australian rainbow trout Oncorhynchus mykiss (Walbaum, 1792) farms. Preventative strategies include increasing water flow and filtration, but when an infection is established, chemical intervention is often required. Formalin (FOR) has been traditionally used on Australian trout farms as a treatment for I. multifiliis. Treatment using sodium percarbonate (SPC) that releases hydrogen peroxide when dissolved is being implemented on a number of farms. To assess anecdotal reports of low efficacy we evaluated 1 h exposures of FOR and SPC at 12 °C and 17 °C in both hard and soft water against free-living stages of I. multifiliis. Each free-living stage were exposed to FOR and SPC in vitro; theronts were exposed to 8, 16, 32 or 64 mg/l SPC or FOR every 15 min, for a maximum of 6 h, and the number of live theronts at each time point was recorded. Prototomonts and tomocysts were exposed to 64, 128, 256 and 512 mg/l SPC and 16, 32, 64 and 128 mg/l FOR for 1 h, incubated, with the percentage viability and the number of theronts produced recorded. Theronts were more sensitive to treatment than tomonts, and prototomonts were more sensitive to treatment than tomocysts. FOR and SPC killed all theronts within 15 min at 64 mg/l at both temperatures. FOR was effective against all prototomonts at ≥64 mg/l at both temperatures and was effective against all tomocysts at 128 mg/l at 17 °C but did not achieve complete mortality in any doses tested at 12 °C. SPC was effective against prototomonts and tomocysts at 64 m/l at 17 °C but required ≥256 mg/l at 12 °C. These results can be used to aid development of specific treatment strategies for the management of I. multifiliis on Australian rainbow trout farms.

Comparison of serum antibody responses and host protection against parasite Ichthyophthirius multifiliis between channel catfish and channel × blue hybrid catfish

There is limited information available on the immune protection of channel catfish Ictalurus punctatus × blue catfish I. furcatus (CB) hybrid against the fish parasite Ichthyophthirius multifiliis (Ich). The objective of this study was to compare serum antibody response and host protection between channel catfish and CB hybrid catfish using a cohabitation model. Channel catfish and CB hybrid catfish were immunized with live theronts by immersion or by IP injection at the dose of 10,000–20,000 theronts per fish in two trials. The fish were then challenged with theronts to compare serum antibody response and protection against the parasite between channel catfish and CB hybrid catfish. The immunized channel catfish and CB hybrid catfish showed a significantly higher (p < 0.05) serum anti-Ich antibody (titer > 1120) compared to non-immunized controls (titer = 0). After being challenged with live theronts, the immunized channel catfish and CB hybrid catfish had none or a low number of the parasites (<50 trophonts per fish) and showed a significantly higher (p < 0.05) survival (90–100%) than non-immunized controls (0%). Overall results indicated that there was no statistical (p > 0.05) difference on serum anti-Ich antibody, parasite infection and fish survival between immunized channel catfish and CB hybrid catfish.

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15–25 °C, 20–25 °C and 20–30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15–25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20–25 °C and 20–30 °C compared to fish at 15 °C, 20 °C and 15–25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0–10.7% mortalities). At cycling water temperature, fish vaccinated at 15–25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20–25 °C and 20–30 °C ( P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20–30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15–25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.

Immune protection of Mozambique tilapia (Oreochromis mossambicus) exposed to different infectious doses of ectoparasite (Cryptocaryon irritans)

The objectives of the present study were to standardize a reproducible infection procedure with Cryptocaryon irritans and to examine the effects of infectious dose level on the immune protection in Mozambique tilapia (Oreochromis mossambicus). This study demonstrated that direct enumeration of trophonts on the pectoral fin was useful to quantitatively assess immune protection against C. irritans. The number of trophonts on a pectoral fin was positively correlated with infectious dose of live theronts. Fish immunized by direct exposure under controlled laboratory conditions allowed for in depth examination of the effects of the degree of infectious dose on immune response. There was no significant positive correlation between the initial infectious dose and degree of immune responses. Mozambique tilapia initiated a strong immune protection by direct exposure with even a small number of parasites (e.g. 300 theronts per fish). Moreover, as the result of the protein analysis, we identified 28kD proteins that could be responsible for the immobilizing antigen.

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: Molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout ( Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 μg protein g −1 fish, 2) 10 trophonts or 20.4 μg protein g −1 fish, 3) 20 trophonts or 40.8 μg protein g −1 fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g −1 fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3–60.0%). The fish immunized with 5 trophonts g −1 fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher ( P < 0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57–77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.

Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.

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.