The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.).

Many studies have shown that coagulation systems play an important role in the defence against pathogens in invertebrates and vertebrates. In vertebrates, particularly in mammals, it has been established that the coagulation system participates in the entrapment of pathogens and activation of the early immune response. However, functional studies investigating the importance of the fish coagulation system in host defence against pathogens are scarce. In the present study, injection of turbot (Scopthalamus maximus) with the pathogenic ciliate Philasterides dicentrarchi led to the formation of macroscopic intraperitoneal clots in the fish. The clots contained abundant, immobilized ciliates, many of which were lysed. We demonstrated that the plasma clots immobilize and kill the ciliates in vitro. To test the importance of plasma clotting in ciliate killing, we inhibited the process by adding a tetrapeptide known to inhibit fibrinogen/thrombin clotting in mammals. Plasma tended to kill P. dicentrarchi slightly faster when clotting was inhibited by the tetrapeptide, although the total mortality of ciliates was similar. We also found that kaolin, a particulate activator of the intrinsic pathway in mammals, accelerates plasma clotting in turbot. In addition, PMA-stimulated neutrophils, living ciliates and several ciliate components such as cilia, proteases and DNA also displayed procoagulant activity in vitro. Injection of fish with the ciliates generated the massive release of neutrophils to the peritoneal cavity, with formation of large aggregates in those fish with live ciliates in the peritoneum. We observed, by SEM, numerous fibrin-like fibres in the peritoneal exudate, many of which were associated with peritoneal leukocytes and ciliates. Expression of the CD18/CD11b gene, an integrin associated with cell adhesion and the induction of fibrin formation, was upregulated in the peritoneal leukocytes. In conclusion, the findings of the present study show that P. dicentrarchi induces the formation of plasma clots and that the fish coagulation system may play an important role in immobilizing and killing this parasite.

A transcriptomic analysis of American lobster (Homarus americanus) immune response during infection with the bumper car parasite Anophryoides haemophila.

Anophryoides haemophila is an important protistan parasite of American lobster, Homarus americanus, as it has been found to infect lobsters in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of over 14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to elucidate molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several anti-lipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms was monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RT-qPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5-7weeks for ALFHa-1 and 10weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during Aerococcus viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health.

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 O(2) 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.

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.

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.

Inonotus obliquus containing diet enhances the innate immune mechanism and disease resistance in olive flounder Paralichythys olivaceus against Uronema marinum.

The present study describes the effect of diet supplementation with Chaga mushroom, Inonotus obliquus extract at 0%, 0.01%, 0.1%, and 1.0% levels on the innate humoral (lysozyme, antiprotease, and complement), cellular responses (production of reactive oxygen and nitrogen species and myeloperoxidase), and disease resistance in olive flounder, Paralichythys olivaceus against Uronema marinum. The lysozyme activity and complement activity significantly increased in each diet on weeks 2 and 4 against pathogen. The serum antiprotease activity and reactive nitrogen intermediates production significantly increased in fish fed with 0.1% and 1.0% diets from weeks 1-4. However, reactive oxygen species production and myeloperoxidase activity significantly increased in 1.0% and 2.0% diets on weeks 2 and 4. In fish fed with 0.1% and 1.0% diets and challenged with U. marinum the cumulative mortality was 50% and 40% while in 0% and 0.01% diets the mortality was 85% and 55%. The results clearly indicate that supplementation diet with I. obliquus at 0.1% and 1.0% level positively enhance the immune system and confer disease resistance which may be potentially used as an immunoprophylactic in finfish culture.

Dietary supplementation with chitin and chitosan on haematology and innate immune response in Epinephelus bruneus against Philasterides dicentrarchi.

The present study investigated the effect of 1.0% chitin and chitosan supplementation diets on haematology and immune response in Kelp grouper, Epinephelus bruneus against protozoan parasite, Philasterides dicentrarchi. The red blood cells (RBC), white blood cells (WBC), haemoglobin levels, lymphocytes, monocytes, and neutrophils significantly increased in kelp grouper fed with chitin or chitosan enriched diets against P. dicentrarchi. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and thrombocytes did not significantly change against pathogen. The phagocytic activity, respiratory burst activity, complement activity, antiprotease activity, and α2-macroglobulin were significantly enhanced in fish fed with 1% chitin and chitosan diet on weeks 2 and 4. The lysozyme activity, total protein, and myeloperoxidase activity significantly increased in fish fed with chitin or chitosan supplementation diet from weeks 1 to 4 against pathogen. The cumulative mortality was found low in fish fed with chitin and chitosan enriched diets than those of control against pathogen. The present study suggests that supplementation of 1.0% chitin or chitosan in diets positively enhances immune response and affords disease resistance in kelp grouper, E. bruneus against P. dicentrarchi infection.

In vivo innate immune responses of groper (Polyprion oxygeneios) against Miamiensis avidus infection and lack of protection following dietary vitamin C administration.

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus, a pathogen of New Zealand groper (Polyprion oxygeneios). The aim of this study was to characterise both the host (groper)-parasite (M. avidus) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO(+) cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus, which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals. Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg(-1)) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo. Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite.

Korean mistletoe enriched diet enhances innate immune response in kelp grouper, Epinephelus bruneus against Philasterides dicentrarchi.

The present study investigated the immunostimulatory effect of Korean mistletoe Viscum album extract (KM-E) on innate immune response in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi. Kelp grouper were divided into four groups of 25 each and fed with 0 (control), 0.5, 1.0, and 2.0% enriched diets with Korean mistletoe extract (KM-E). After feeding for 30 days, the fish were injected intraperitoneally (i.p.) with 100 µl of P. dicentrarchi (4.2 × 10(7)ciliates/ml) to study the immune responses at weeks 1, 2, and 4. The respiratory burst activity did not significantly enhance when fed with 0.5% and 1.0% supplementation diets on week 1 when compared to control diet. On weeks 2 and 4, the respiratory burst activity significantly increased with 1.0% and 2.0% diets. The phagocytic activity significantly enhanced with 1.0% and 2.0% diets, but not with 0.5% diet at any time. When fed with 1.0% KM-E-diet the lysozyme activity did not significantly vary at any week whereas with 1.0% and 2.0% diets it was significantly enhanced. The total protein level significantly increased with 1.0% and 2.0% KM-E-diets from weeks 1 to 4 as compared to control. The present study suggests that 1.0% or 2.0% KM-E-supplementation diet positively enhances the innate immune response in E. bruneus against P. dicentrarchi infection.

Turbot resistance to Philasterides dicentrarchi is more dependent on humoral than on cellular immune responses.

Philasterides dicentrarchi is a ciliate that causes high mortalities in cultured turbot, Psetta maxima (L.). This pathogen displays high phagocytic activity and after entering the body it multiplies and feeds on host cells and tissue components. In previous studies, we found that complement, activated through the classical pathway, is a potent killer of P. dicentrarchi. Here, we compared the killing activity of turbot leucocytes and humoral factors against two virulent isolates of P. dicentrarchi, in order to determine the importance of leucocytes in the defence against this pathogen. Components of P. dicentrarchi (ciliary and membrane) stimulated turbot leucocytes, and increased the respiratory burst, degranulation and the expression of pro-inflammatory cytokines. We tested the susceptibility of ciliates to reactive oxygen and nitrogen species, by incubating them with different oxidative systems (H(2)O(2), Fe/ascorbate, which induces lipid peroxidation, an O(2)(-) donor (XOD/HX), an NO donor (SNAP) and an ONOO(-) donor (SIN-1)), for 24h. Both isolates were susceptible to high concentrations of H(2)O(2,) Fe/ascorbate, XOD/HX, and SIN-1 but were resistant to incubation with SNAP. Leucocytes became strongly activated when they were in contact with or were phagocytosed by the ciliate. Incubation of P. dicentrarchi with a combination of fresh serum and specific antibodies killed most of the ciliates, but the addition of leucocytes to ciliate cultures did not increase the toxicity to the ciliates. On the contrary, the number of ciliates increased when leucocytes were added to the culture because the ciliates fed on them. Despite being activated, leucocytes did not produce sufficiently high concentrations of toxic substances to kill the parasite. The most virulent isolate was that which induced greatest activation of leucocytes but was least susceptible to complement. We concluded that humoral factors such as complement (activated through the classical pathway) are critical for fish defence against P. dicentrarchi and that cellular responses appear to play a minor role, if any, in defence against this ciliate.

Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder.

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40+/-3.1% to 79.4+/-1.7%) and it decreased from August to November (63+/-2.3% to 30+/-2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49+/-7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100ppm for 30min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.

Antigenic differences of the scuticociliate Miamiensis avidus from Japan.

In Japan and Korea, outbreaks of scuticociliatosis have frequently occurred in Japanese flounder, Paralichthys olivaceus. Morphological observations and small subunit rRNA gene sequences have shown that the causative agent of scuticociliatosis in the flounder is Miamiensis avidus (syn. Philasterides dicentrarchi). In this study, we elucidated the antigenic differences between six Japanese M. avidus isolates as an initial step toward developing an effective vaccine against the disease. Four Japanese flounder isolates (IyoI, Nakajima, JF05To and Mie0301 isolates), one spotted knifejaw, Oplegnathus punctatus, isolate (SK05Kyo), and one ridged-eye flounder, Pleuronichthys cornutus, isolate (RF05To) were subjected to serological analysis. Antisera against IyoI, SK05Kyo, Nakajima and Mie0301 isolates were raised in rabbits and used for immobilization assays and Western blotting. Immobilization assays showed that the six isolates could be divided into three groups, tentatively designated serotype I for IyoI, JF05To, RF05To, SK05Kyo, serotype II for Nakajima and serotype III for Mie0301. Western blotting results supported these three serotypes, with marked similarities in the banding profiles of IyoI, JF05To, RF05To and SK05Kyo isolates, which were distinct from the Nakajima and Mie0301 isolates. Three isolates, IyoI, Nakajima and Mie0301 that were selected as representatives of each serotype, were highly pathogenic to Japanese flounder by experimental infection. Based on these findings, we propose that there are at least three M. avidus serotypes in Japan.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection.

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.

Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C.

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.

Philasterides dicentrarchi (Ciliophora:Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS-PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.