A vaccine based on biodegradable microspheres induces protective immunity against scuticociliatosis without producing side effects in turbot

The histiophagous scuticociliate parasite Philasterides dicentrarchi is an emergent pathogen in aquaculture and causes significant economic losses on turbot (Scophthalmus maximus) farms. In this study, the surface antigens (Ag) of the parasite were encapsulated and covalently linked to a polymeric microparticle formulation composed of two biodegradable polymers (chitosan and Gantrez). The antigenicity of the formulation and the protection provided were compared in mice and turbot. This formulation induced a higher antibody (Ab) response in mice at doses of 5mg of microspheres (MS) conjugated with approximately 230 μg of Ag (MS-Ag(c)). However, Ab levels were significantly lower than in mice vaccinated with the same concentration of Ag in complete Freund's adjuvant (FCA). In turbot, the MS-Ag(c) formulation induced a higher level of Abs than that induced by the same vaccine emulsified in FCA. The challenge experiments performed with P. dicentrarchi and vaccinated turbot also showed a clear correlation between Ab levels and survival levels. Growth was significantly affected in fish vaccinated with FCA, but not in fish vaccinated with MS. The high adjuvant capacity of MS, together with its biodegradability and low toxicity to fish, makes this new vaccine an economical, effective and safe alternative to oil-based adjuvants for the immunoprophylaxis of scuticociliatosis in turbot.     

Poly D,L-lactide-co-glycolic acid (PLGA)-encapsulated vaccine on immune system in Epinephelus bruneus against Uronema marinum

We investigate the efficacy of poly D,L-lactide-co-glycolic acid (PLGA)-encapsulated vaccine on innate and adaptive immune response in kelp grouper (Epinephelus bruneus) against Uronema marinum at weeks 1, 2, and 4. The respiratory burst (RB) activity, complement activity, and α2-macroglobulin were significantly enhanced in fish immunization with vaccine on week 4 whereas vaccine and PLGA-encapsulated vaccine from weeks 1 to 4. The serum lysozyme activity, antiprotease activity, and antibody level were significantly enhanced in fish immunized with vaccine and PLGA-encapsulated vaccine on weeks 2 and 4. The cumulative mortality was low in PLGA-encapsulated vaccine with 20% whereas high in PLGA and vaccine with 40% and 30%. The results from the present study suggest that PLGA-encapsulated vaccine is useful for further design of immunoprophylatic nano formulation against scuticociliatosis.     

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.     

Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae

A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5x10(4)CFUS. agalactiae fish(-1). Blood was drawn from all fish 90d after vaccination and 25d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72h later with 1.5x10(4)CFUS. agalactiae fish(-1), and significantly less (P<0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0-10.0% mortalities) than in control groups (63.3-72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P<0.9455) or vaccinated-challenged fish (P<0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P<0.0001) specific antibody levels over control fish. A highly significant (r(2)=0.5892; P<0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish.     

The activity of several components of the innate immune system in diploid and triploid turbot

The use of triploid fish may be of interest in research, e.g. study of how this condition affects the size and activity of cells. In addition, triploid fish are sterile and production of triploids in fish species that are marketed after reaching sexual maturity may be of economic interest. In the present study, the effects of triploidy on the activity of several components of the innate immune system of turbot (Psetta maxima L) were determined. Triploid turbot had bigger cells (erythrocytes and neutrophils) but the number of blood erythrocytes, leucocytes and thrombocytes was lower than in diploid fish. The differential cell count was similar in both types of fish. The respiratory burst and the phagocytic activities were higher in neutrophils of triploid turbot. However, because the number of neutrophils was higher in diploids, the total respiratory burst activity and the phagocytosis per microliter of blood was similar in both types of fish. No differences were found in serum complement, lysozyme or bactericidal activities. The results indicate that the activities of the humoral components of the innate immune system tested are similar in diploid and triploid fish and that the lower leucocyte number found in triploids is compensated for by higher cell activity.     

Effects of the histiophagous ciliate Philasterides dicentrarchi on turbot phagocyte responses

Philasterides dicentrarchi is an opportunistic histiophagous ciliate parasite causing systemic scuticociliatosis in cultured turbot (Scophthalmus maximus L.). This study investigated the effects of inoculation with live or killed trophozoites of this ciliate (plus 3% thioglycollate) on the in vitro phagocytic activity and respiratory-burst responses of inflammatory peritoneal leucocytes obtained from the fish thus treated. The phagocytic activity of leucocytes from fish inoculated with killed P. dicentrarchi was higher in the presence than in the absence of infected turbot serum (ITS). The effect of ITS was smaller in fish inoculated with live P. dicentrarchi, indicating modulation of the opsonic activity of ITS. Inoculation with live ciliates led to a significant increase in subsequent in vitro extracellular ROS production, but only when normal turbot serum (NTS) or ITS was included in the assay medium. Inclusion of live P. dicentrarchi in the medium abolished this increase, suggesting ROS-scavenging activity. Inoculation with live P. dicentrarchi led to a significant decline in subsequent in vitro intracellular ROS production; when NTS was included in the medium, there was a significant increase in intracellular ROS production, but no such increase was observed when ITS was included in the medium. Inoculation with live P. dicentrarchi alone did not increase subsequent in vitro NO? production in response to LPS; a significant increase was observed when NTS or ITS was included in the assay medium, but this increase was not affected by prior inoculation with P. dicentrarchi. These results suggest that the amphizoic nature of this parasite may reflect the ease with which it can develop mechanisms of evasion of the host immune response.     

Plasmodium chabaudi: Humoral and cell-mediated responses of immunized mice

Antigen preparations of Plasmodium chabaudi parasites enriched in merozoites and schizonts, obtained from in vitro culture, and combined with saponin protected C57BL/6J mice from P. chabaudi infection as judged by reduced primary parasitemias and recrudescences. Sera passively transferred from immunized and untreated mice after a challenge infection were more protective in recipients than serum from normal mice. Mice treated with antilymphocyte serum during immunization did not develop as strong an immunity to infection as did controls treated with normal serum. Immunized mice had depressed delayed-type hypersensitivity reactions to malarial antigen but increased serum titers of malarial antibody (measured by imniunofluorescence) after challenge with P. chabaudi when compared to immunized mice which remained unchallenged. The protective activity of sera from various groups of mice did not necessarily correlate with the serum antibody titers.     

The immune response of carp, Cyprinus carpio L., following injection of antigen-antibody complexes

Cyprinus carpio immunized with antigen-antibody complexes formed at equivalence were found to have both good memory and enhanced serum antibody responses compared with fish primed with antigen alone. Only priming with antigen or complex in adjuvent produced comparable results. Complexes formed in antigen excess gave better precipitation responses during the secondary response than in corresponding control groups injected antigen alone or in adjuvant. The role of antigen-antibody complexes in the fish immune system and the possibility of using such complexes to vaccinate fish are discussed.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21-28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

Abstract We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21–28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

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.     

Characteristics of protective immunity in mice induced by drug-attenuated larvae of Schistosoma mansoni. Antigen localization and antibody responses

Partial resistance to reinfection developed in mice with immature infections of Schistosoma mansoni treated with the drug Ro11-3128, whereas mice vaccinated with irradiated cercariae and treated in a similar way did not become immune. Persistence of drug-treated parasites in the skin and draining lymph nodes, which prolonged the opportunity for efficient Ag presentation, was necessary for the development of protective immunity. Death and clearance of parasites solely in the skin, was not sufficient to induce protection. The expansion in the number of lymphocytes in the skin-draining nodes after vaccination, was reflected in the contrasting levels of resistance induced by the different drug-treatment regimes. Challenge parasites were eliminated predominantly after they reached the lungs. An investigation of antibody reactivity revealed an immunodominant response against a doublet of Ag of Mr 97 to 99 kDa. Recognition of this complex by antisera from different groups of mice was not related to their immune status. Western blots and inhibition analysis showed that this doublet has epitopes in common with the Sm97/paramyosin protective Ag, originally identified by antisera reactivity from mice immunized with a nonliving vaccine.     

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain)

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.     

Biodegradable microparticles covalently linked to surface antigens of the scuticociliate parasite P. dicentrarchi promote innate immune responses in vitro

The histiophagous scuticociliate endoparasite Philasterides dicentrarchi is an emerging pathogen that infects the turbot Scophthalmus maximus and thus causes important economic losses in turbot aquaculture. This in vitro study investigated the adjuvant capacity of biodegradable microspheres (MS) composed of two polymers (chitosan and Gantrez^®) covalently coupled to surface antigens (Ag) of P. dicentrarchi. The coupled MS–Ag significantly stimulated the phagocytic response of both murine macrophages (RAW 264.7 cells) and leukocytes from the anterior kidney of turbot (HLK), at a level similar to that induced by zymosan A. The MS–Ag also significantly increased production of reactive oxygen and nitrogen species, as shown by the increased O₂ consumption and stimulation of the respiratory burst and nitric oxide production by murine and in particular by turbot HLK. The MS–Ag stimulated the production of the proinflammatory cytokine tumour necrosis factor alpha (TNFα) by murine and turbot HLK. The results confirm the high adjuvant capacity of biodegradable MS covalently bound to Ag as regards stimulating the innate immune response, and they justify the use of MS in the production of safe and effective vaccines against fish pathogens.     

Plasmodium fragile: Inhibition of cultures by serum from Rhesus monkeys immunized with homologous parasites

Rhesus monkeys, Macaco mulatto, that had previously been immunized with the Nilgiri strain of Plasmodium fragile grown in culture, together with control monkeys with and without inoculation of Freund's adjuvant, were challenged with cultured parasites. After treatment with chloroquine, the monkeys were rechallenged. Serum specimens from three immunized monkeys caused a specific, dose-dependent inhibition of parasite growth in culture. Fifty percent inhibition of in vitro growth was obtained using 5% immune serum combined with 10% normal rhesus serum. The specific inhibitory component of immune serum was shown to be IgG antibody. Results of the study demonstrated that there is good correlation between the inhibitory activity of immune serum, parasite growth in vitro, the in vivo response to challenge, and the indirect fluorescent antibody titer.     

卵形鲳鲹对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫（Cryptocaryon irritans）的幼虫对卵形鲳鲹（Trachinotus ovatus）进行腹腔注射和体表感染,然后每隔1周用阻动试验（Immobilization assay）检测免疫鱼的抗血清和皮肤培养液对激刺隐核虫幼虫的阻动效价,在第14周,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示：两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异：两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤粘膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是粘膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。