The immune response of the thick-lipped grey mullet, Chelon labrosus (Risso, 1826), to metacercarial infections of Cryptocotyle lingua (Creplin, 1825)

Grey mullet, Chelon labrosus , (60–100 g) have been found to respond immunologically to Cryptocotyle lingua , following a single exposure to 20000 cercariae, by the production of humoral antibody, sensitized pronephric leucocytes and cytotoxic serum factors. Antibody titres measured by passive haemagglutination reached a peak at week 4 with a -log₂ titre of 16±S.E. 1.0, and titres of 10.7±S.E. 1.0 were still recorded after 10 weeks at the termination of the experiment. Cercarial agglutination was found unreliable as a rapid test. Pronephric leucocytes, sensitive to cercarial antigen when measured by the under-agarose migration method, were detected between weeks 1 and 6, peaking at week 2. In vitro polarization was increased when cells were incubated with the antigen, but this increase was not significantly different between control and infected fish. Heat-labile cytotoxic factors of immune sera have been demonstrated to whole cercariae in vitro , these factors being associated with structural damage to the tegument. Pronephros cells isolated from immune fish during each week of infection showed no evidence of adherence to cercariae or metacercariae in vitro. The results are discussed in relation to both host response and course of infection of the parasite.     

Composition of leucocytes of the head kidney of the crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>, Cypriniformes: Cyprinidae) as affected by invasion of cestode <Emphasis Type="Italic">Digramma interrupta</Emphasis> (Cestoda; Pseudophyllidea)

The composition of leucocytes of the head kidney is studied in the crucian carps (Carassius auratus) either contaminated or uncontaminated with Digramma interrupta. The composition of leucocytes in the pronephros of the crucian carp from Lake Baikal basin has a lymphoid character. Compared to the crucian carp from the European part of Russia, in the fish from Baikal the granulocytopoetic processes are more pronounced. This is proved by the high content of young forms of granulocytes. In the fish infected with digramma, the immune suppression of proliferation of blasts and young forms of eosinophils was revealed. On the other hand, the inflammatory and humoral specific immune reactions are enhanced. Partial suppression of the immune response of C. auratus to invasion by D. interrupta facilitates development of the parasite.     

Humoral immune response of carp Cyprinus carpio to Myxobolus artus (Myxozoa: Myxobolidae) infection

The circulating antibody which reacted with sonicated spores of Myxobolus artus was detected in some naturally infected carp. However, some other fish had no detectable sign of infection, though they had the antibody. When carp were injected either with intact or sonicated spores, the antibody was not produced, while fish injected either with developing stages (presporogonic and sporoblast stages) of the parasite or sonicated spores with bovine serum albumin elicited the antibody production. The results of the injection experiments suggest that (1) developing stages have antigenicity to carp, and (2) spores have lost the antigenicity; sonicated spores are haptens, with which the antibody can react. In an indirect fluorescent antibody technique, sera positive for the antigen reacted with developing stages of the parasite, but not with the spore.
The mechanism of the host immune response against M. artus is discussed in relation to a previous observation that the parasite sometimes underwent abnormal development, in which host encapsulation was imperfect or even lacking, probably leading to degeneration of pseudocysts before the completion of spore formation. It is plausible that the antibody was produced when pseudocysts which showed abnormal growth ruptured during their developing stages, resulting in exposure of the young parasite to the host immune system.     

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.     

Cadmium-induced cellular and immunological responses in Cyprinus carpio infected with the blood parasite, Sanguinicola inermis

Little is known about immune responses in teleosts as linked to the aetiology of pollutants and parasitic diseases and in particular their combined effects on the host. Cadmium(Cd)-mediated immunological responses in the thymus and pronephros of juvenile carp (Cyprinus carpio), experimentally infected with the blood parasite, Sanguinicola inermis (Trematoda: Sanguinicolidae) for 30 days followed by an exposure to 0.1 mg Cd2+ l(-1) for 48 or 168 h were investigated. Differential organ-specific changes occurred in both organs examined. In carp exposed to Cd, intracelluar spaces, vacuolation in the eosinophils, dissociation of cell membranes together with the formation of concentric whorls occurred. The thymus of infected carp exposed to Cd had a granular cytosol which contained vesicles with electron-dense inclusions, swollen mitochondria with distended cristae and condensed nuclei in the erythrocytes. Cell counts on the two organs revealed a differential response to cadmium exposure in S. inermis infected carp compared to control infected fish. A significant increase in the neutrophil, eosinophil and thrombocyte components occurred in the thymus in contrast to a significant decrease in pronephric neutrophils. In addition, there was a differential blastogenesis response in infected and Cd-exposed infected carp fry exposed to cercarial antigens and the mitogens, concanavalin A and pokeweed mitogen.     

Susceptibility of carp, roach and goldfish to a Dermocystidium-like organism

A visceral form of a Dermocystidium -like organism was found at 13% prevalence in a carp and in 7·4% in a roach population from Greece. Successful experimental transmission was recorded only in carp. Pathology of the parasite in experimental fish and naturally infected fish was similar. ©1998 The Fisheries Society of the British Isles     

In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against cryptobiosis in Oncorhynchus mykiss

The present communication reports on the attenuation of a pathogenic hemoflagellate, Cryptobia salmositica Katz (Sarcomastigophora: Kinetoplastida) and its use as a live vaccine against cryptobiosis. The parasite was attenuated by continuous in vitro culture (at 10 C for 55 wk) in minimum essential medium. Attenuated (culture) forms are morphologically similar to virulent (blood) forms. They are however more slender and have a shorter anterior flagellum and a smaller nucleus and kinetoplast. The attenuated form returned to its normal size and multiplied when inoculated into naive Oncorhynchus mykiss. It produced a low parasitemia but did not cause disease (e.g., no exophthalmia or anemia) in fish. At four wk after infection, the vaccinated fish were challenged with the virulent parasite. They were protected from the disease, whereas the control (naive) fish, infected with only the virulent parasite, had the usual clinical signs (e.g., anemia, exophthalmia). No parasite was detected in any of 10 vaccinated fish at 22 wk after challenge with the virulent parasite. However, 5 of 9 fish infected with culture forms and 6 of 9 fish infected with blood forms still had detectable parasites at 26 and 22 wk after infection, respectively.     

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide,which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.     

Head kidney neutrophils of carp (Cyprinus carpio L.) are functionally modulated by the haemoflagellateTrypanoplasma borreli

In the present work responses of carp (Cyprinus carpio) head kidney-derived neutrophils to the blood parasite T. borreli, and the consequences of these responses for parasite survival and other host response mechanisms, were studied. In co-cultures of head kidney leucocytes (HKL) with viable and lysed T. borreli a prominent shape change of neutrophilic granulocytes towards increased size and complexity was observed. In addition, the longevity of neutrophils in vitro was prolonged in the presence of T. borreli antigens. In these cultures, neutrophils also exhibited an increased phagocytosis activity. An up regulation of the production of nitric oxide (NO) and reactive oxygen species (ROS) was observed in T. borreli- and mitogen-stimulated HKL cultures. However, addition of live, fluorescence-labelledT. borreli to previously stimulated HKL cultures, revealed neither killing nor phagocytosis of the parasite by activated neutrophils. Moreover, viable T. borreli, when added to HKL cultures of infected carp, reduced their phagocytosis activity and NO production. Supernatants of co-cultures between T. borreli and HKL also contained mediators, which suppressed a mitogen-induced proliferative response of peripheral blood leucocytes (PBL) in vitro. Thus, while T. borreli itself appeared not to be sensitive to responses of activated neutrophils, the flagellates interferes with the production of immunomodulatory signals of these cells, probably resulting in a partial immunosuppression, which may favour the parasite development in vivo.     

Rainbow trout leucocyte activity: influence on the ectoparasitic monogenean Gyrodactylus derjavini

The ectoparasitic monogenean Gyrodactylus derjavini from rainbow trout Oncorhynchus mykiss was exposed in vitro to macrophages isolated as peritoneal exudate cells or as pronephros cells from the host. Cells colonized the parasite especially in the mannose-rich regions in the cephalic ducts where ciliated structures were abundant. Opsonization with fresh serum, in contrast to heat-inactivated serum, enhanced colonization also on other body parts. The adverse effect of the activated macrophages towards G. derjavini was associated with a heat-labile component released from these cells to the culture medium. Analysis of substances released from the cells showed reactivity for a number of enzymes, complement factor C3, interleukin (Il-1) and reactive oxygen metabolites. Chemotaxis assays with pronephric leucocytes showed chemoattractants in G. derjavini, and the respiratory burst level of macrophages was slightly elevated due to parasite exposure. It is suggested that skin leucocytes contribute to an increased level of complement factors in the trout skin during the host response, whereby a hostile microenvironment for the parasites is created. In addition, the IL-1 production could affect mucous cell secretion and hyperplasia and add to the antiparasitic action of the epithelium. Likewise, reactive oxygen metabolites and various enzymes are likely to be involved in the skin response.     

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM).Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.     

Immunization of rainbow trout Oncorhynchus mykiss against Discocotyle sagiffata (Monogenea)

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with 2 different Discocotyle sagittata extracts dissolved in PBS and subsequently exposed to controlled infection. Immunization resulted in significantly reduced (p < 0.0001) worm intensities in > 50% of vaccinated fish (response arbitrarily defined as parasite burdens < mean control intensity - 1 SD), irrespective of the immunization regime (different parasite extracts, dosing and application schedules) employed. The protective effect of worm extract applied in Freund's complete adjuvant (FCA) did not differ significantly from extract given in PBS. Vaccination with embryonated parasite eggs extract and with FCA alone did not result in partial immunity, suggesting the observed protective effect is specific. Immunized fish had significantly higher specific antibody titres at the time of dissection (as determined by ELISA) than both naive and control fish. Overall, a significant negative correlation was found between antibody titres and worm burdens, suggesting immunoglobulins are implicated in mediating partial immunity. Western blot tests indicated the 2 different worm extracts used to immunize fish share antigens, but each one primarily induced recognition of a distinct band (30 and 38 kDa). Immunization seems to promote a shift between 2 equilibria, rather than progressively increasing protection. This would explain why boosting did not increase immunity, and why 2 different extracts primarily inducing recognition of 2 distinct antigens provide similar degrees of protection. Although several other non-specific and cellular factors are likely to be involved in controlling parasite numbers, it cannot be excluded that antibodies could be involved in mediating the observed partial immunity.     

Efficacy of quinine against ichthyophthiriasis in common carp Cyprinus carpio

Ichthyophthiriasis, caused by Ichthyophthirius multifiliis, is an economically important worldwide parasitic disease that infects all freshwater fish. Since the banning of malachite green for use in food fish, there has been a great need for alternative therapeutants. The goal of this study was to investigate the efficacy of quinine against I. multifiliis. Parasite developmental stages from our laboratory-established life cycle in rainbow trout Oncorhynchus mykiss were exposed to quinine in vitro, and a dual fluorescent staining technique was used to allow a clear distinction between viable and damaged parasites. Furthermore, the effect of quinine was assessed in vivo by oral administration and intraperitoneal injections in common carp Cyprinus carpio. The results of the in vitro experiments proved quinine to be effective against the parasite. Quinine injected at a dosage of 60 mg kg(-1) body weight resulted in a significantly lower number of trophonts. In contrast, in-feed trials did not show a significant reduction of trophonts after treatment commencing 1 d after infection with concentrations of up to 20 g quinine kg(-1) feed for 3 d. After a 14-d treatment at concentrations of up to 10 g quinine kg(-1) feed prior to theront exposure, there was also no significant difference in parasite numbers between treated and control groups. The results of oral versus parenteral application of quinine indicate that the substance is not completely absorbed from the intestinal tract of common carp. However, medicated feed containing higher concentrations of quinine was less readily accepted by the fish, presumably due to the bitter taste.     

Specific IgE induced by Kudoa sp. (Myxosporea: Multivalvulida) antigens in BALB/c mice

The majority of Kudoa species infect the somatic muscle of fish establishing cysts. As there is no effective method to detect infected fish without destroying them, these parasited fish reach the consumer. We have developed this work to determine whether this parasite contains antigenic compounds capable of provoking an immune response in laboratory animals, in order to consider the possible immunopathological effects in man by the ingestion of Kudoa infected fish. BALB/c mice were injected by the subcutaneous route with the following extracts suspended in aluminium hydroxide: Group 1 (black Kudoa sp. pseudocyst extract), group 2 (white Kudoa sp. pseudocyst extract). Specific IgE levels were measured by ELISA. IgE detected in both groups 1 and 2 showed the possible allergenic nature of some of the components of the parasitic extracts.     

Modulation of the mitogenic PHA response of carp, Cyprinus carpio L., by extracellular products of Aeromonas salmonicida

The influence of bacteria-free supernatants from cultures of atypical virulent (V234/81, auto-agglutinating. A-layer positive) and avirulent (126/68, non-agglutinating, A-layer negative) strains of A. salmonicida , obtained after different culture times in yeast-tryptone broth at 20°C, was tested on the PHA response of carp pronephric leucocytes in vitro . Supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The response was enhanced (400%) by supernatant from early virulent cultures (20 h), but severely depressed (<3%) by later ones (96 h). The effects were dose-dependent. Inhibitory activity of 96-h supernatant was lost by heating (70°C, 30 min), suggesting that the inhibiting factors are all proteinaceous.
Heated 96-h supernatant was as stimulatory as early supernatant. Stimulation of leucocytes also occurred in the absence of PHA with early and heat-treated 96-h supernatants, but at a tenth of the level, suggesting that only stimulated cells (blasts) might respond to the substance(s) present in supernatants. Membrane fragments from virulent and avirulent bacteria, and purified LPS from virulent bacteria, were stimulatory with or without PHA. Endotoxin-free, heat-treated, 96-h culture supernatants were also stimulatory, suggesting that an additional mitogenic factor(s), other than LPS, is present in the supernatants. The modulating in vitro effects of extracellular products from A. salmonicida might explain the immunosuppression seen during later stages of erythrodermatitis in vivo.     

Modulation of granulocyte responses in three-spined sticklebacks Gasterosteus aculeatus infected with the tapeworm Schistocephalus solidus

Leukocytes isolated from the head kidney and peripheral blood of 3-spined sticklebacks Gasterosteus aculeatus L. were analysed by means of flow cytometry during infection with the tapeworm Schistocephalus solidus (Müller, 1776). Although parasites increased their body weight continuously throughout the observation period (98 d), proportions of granulocytes increased in blood and head kidney only up to Day 63 post-infection (p.i.). Thereafter, declining proportions of granulocytes were observed in both organs. Thus the relative decrease in granulocyte number was not correlated to a decline in the parasitic load of the fish. To investigate a possible modulatory impact of S. solidus on granulocyte function, head kidney leukocytes were isolated at times before Day 63 p.i. and tested in vitro for their capacity to produce reactive oxygen species (ROS). Head kidney leukocytes from S. solidus-infected fish, analysed immediately after isolation (ex vivo, Day 40 p.i.), exhibited a higher ROS production when stimulated with phorbol myristate acetate (PMA), than leukocytes from naive, sham-treated control fish and fish that had resisted or cleared the infection (exposed but not infected). The latter showed an increased spontaneous ROS production that was not correlated to the numbers of granulocytes present in the head kidney isolates. In infected sticklebacks, spontaneous and PMA-induced ROS production was significantly correlated with numbers of granulocytes present in the head kidney isolates, suggesting that elevated ROS production was due to higher numbers of responding cells rather than an increased capacity of single cells. In vitro, after cultivation for 4 d in the presence of pokeweed mitogen (PWM) or extracts from S. solidus, head kidney leukocytes from control fish showed an increased ROS production and phagocytic activity compared with non-stimulated control cultures. In contrast, head kidney leukocytes from infected fish isolated on Days 48 and 44 p.i., failed to respond to S. solidus antigens in vitro. During S. solidus infection, granulocyte mobilisation resulted in elevated numbers of these cells in head kidneys, but the lack of an in vitro response to S. solidus antigens indicates an in vivo priming of granulocytes by the parasite. These observations may reflect the ability of S. solidus to impair the host's immune response once the parasite is developing in the body cavity of G. aculeatus.     

Scuticociliate cysteine proteinases modulate turbot leucocyte functions

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 microg ml(-1), and it was found that the viability of the leucocytes was not affected after treatment for 24h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2(-)). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, beta-glucans (100 microg ml(-1)) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 microg ml(-1) of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml(-1), and the addition of low levels of PGE2 (62.5 pg ml(-1)) to PL cultures stimulated O2(-) production, although addition of PGE2 at concentrations higher than 250 pg ml(-1) blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1beta. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.     

Schistosoma mansoni: schistosomulicidal activity of macrophages isolated from liver granulomas of infected mice

Mice infected with Schistosoma mansoni develop T cell-mediated granulomatous reactions around disseminated parasite eggs. In this study, granuloma-derived leucocytes have been examined for schistosomulicidal capacity by the use of in vitro cytotoxicity assays. Adherent macrophages, that were shown by electron microscopy to exhibit no gross morphological abnormalities, were unable to mediate significant mortality in the absence of serum factors. When cocultured with immune serum and complement, however, these cells killed +/- 26% of the larvae at a cell:target ratio of 5000:1. In contrast, granuloma-derived cell populations that were enriched for eosinophils (50-70% eosinophil content) showed only minimal cytotoxic potential. This may be related to observed structural changes in the eosinophil lysosomal granules, or perhaps to blocking of the cell-surface receptors by immune complexes. It is concluded that granuloma macrophages, activated by egg antigen-sensitised T lymphocytes, may serve as effector cells in immunity to schistosomules.     

Immunomodulatory effects of secondary metabolites from thermophilic Anoxybacillus kamchatkensis XA-1 on carp, Cyprinus carpio

A bacterial strain with putative immunomodulatory properties was isolated from Xi'an hot springs in China. Comparison of 16S rRNA gene revealed a 97% similarity between the tested strain (designated XA-1) and Anoxybacillus kamchatkensis. Two compounds isolated from the secondary metabolites of XA-1 were identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) cyclo (Gly-L-Pro) and (2) cyclo (L-Ala-4-hydroxyl-L-Pro). Two cyclic dipeptides showed stimulatory properties towards a range of parameters when a dose of 20mg kg(-1) body weight was intraperitoneally injected in naive common carp, Cyprinus carpio. Innate immune parameters (serum SOD, lysozyme and bactericidal activity, and phagocytic activity by peripheral blood leucocytes) along with the expression of two immune-related genes (IL-1β and iNOS) in blood were examined after 7, 14, 21, and 28 days of injection. In the absence of infection, immunomodulators should ideally not affect normal physiology and immunity of the host; possible negative outcomes of activated immune responses in the naive state are discussed. Protection by two bacterial dipeptides was assessed in an intraperitoneal injection challenge trial with live Aeromonas hydrophila. Both compounds reduced mortality, with the highest survival rate observed in the group that received compound 2 (80%) followed by the group that received compound 1 (65%) while control group scored the worse (15%). Elucidation of the involved protective mechanisms in carp requires future studies.     

Detection of Myxobolus rotundus (Myxozoa: Myxosporea) in skin mucus of crucian carp Carassius auratus auratus using a monoclonal antibody

Diagnosis of myxosporean Myxobolus rotundus infection was conducted by examining skin mucus from the infected crucian carp Carassius auratus auratus with a monoclonal antibody, MAb 2D12, raised previously against the parasite. A positive reaction was observed in skin mucus collected from infected fish, and spores and pre-spore stages of the parasite were identified by the MAb 2D12. It was also demonstrated that M. rotundus infection can be successfully detected by a simple method, enzyme-linked immunosorbent assay (ELISA), and that skin mucus collected from infected fish skin had a significantly higher optical density (OD) value than that from uninfected fish.