In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes.

The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as ß-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated ß(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1-150 µgml-1. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25-250 µgml-1 in comparison to control (sterile water), cellulose ether, MacroGard® and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 µgml-1. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1ß), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.

Time and concentration dependency of MacroGard® induced apoptosis.

In previous studies an effect of ß-glucan on apoptosis in fish was noted and in this investigation we determine the time and concentration dependency of this effect. Primary cell cultures of pronephric carp cells were incubated for 6, 24, 48 h with various concentrations ranging from 0 to 1000 µg/ml of MacroGard(®) ß-glucan. Apoptosis was monitored via acridine orange staining. Results indicate a clear effect of time and concentration on the induction of apoptosis in vitro, with only concentration ≥500 µg/ml causing significantly higher percentages of apoptotic cells. Apoptosis was detected after 6 h. This concentration dependent effect has to be considered when studying apoptosis in relation to immunostimulation.

Oxidative burst and nitric oxide responses in carp macrophages induced by zymosan, MacroGard(®) and selective dectin-1 agonists suggest recognition by multiple pattern recognition receptors.

ß-Glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing ß-glucans, with the C-type lectin receptor dectin-1 in conjunction with Toll-like receptor 2 (TLR2), considered key receptors for recognition of ß-glucan. In our studies to determine the possible involvement of these receptors on carp macrophages a range of sources of ß-glucans were utilized including particulate ß-glucan preparations of baker's yeast such as zymosan, which is composed of insoluble ß-glucan and mannan, and MacroGard(®), a ß-glucan-based feed ingredient for farmed animals including several fish species. Both preparations were confirmed TLR2 ligands by measuring activation of HEK293 cells transfected with human TLR2 and CD14, co-transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. In addition, dectin-1-specific ligands in mammals i.e. zymosan treated to deplete the TLR-stimulating properties and curdlan, were monitored for their effects on carp macrophages by measuring reactive oxygen and nitrogen radicals production, as well as cytokine gene expression by real-time PCR. Results clearly show the ability of carp macrophages to strongly react to particulate ß-glucans with an increase in the production of reactive oxygen and nitrogen radicals and an increase in cytokine gene expression, in particular il-1ß, il-6 and il-11. We identified carp il-6, that was previously unknown. In addition, carp macrophages are less, but not unresponsive to selective dectin-1 agonists, suggesting recognition of ß-glucans by multiple pattern recognition receptors that could include TLR but also non-TLR receptors. Candidate receptors for recognition of ß-glucans are discussed.

The influence of dietary ß-glucan, PAMP exposure and Aeromonas salmonicida on apoptosis modulation in common carp (Cyprinus carpio).

The association between ß-glucan (MacroGard®) supplemented feed and apoptosis in immune-related organs of common carp (Cyprinus carpio) was studied using fluorescence microscopy and real-time PCR. In addition the effect of Aeromonas salmonicida, LPS and Poly(I:C) injections on this relationship was evaluated. Whilst acridine orange staining revealed that apoptosis levels were independent of MacroGard® and LPS/Poly(I:C) administration or their combination, it was shown that injection with A. salmonicida increased the percentage of apoptotic cells irrespective of the feeding regime. It was apparent that in all the treatments gene expression profiles displayed organ and time dependency. For example no effect was observed at 7 days of MacroGard® administration while 25 days of feeding led to increased iNOS expression and differential up-regulation of anti- or pro-apoptotic genes depending on organ. This may indicate differences in NO sensitivity. MacroGard® also led to an elevation of pro- as well as anti-apoptotic genes in LPS or Poly(I:C) injected fish, while LPS/Poly(I:C) alone had little effect. A. salmonicida caused enhanced iNOS expression and it is possible that the type of apoptosis pathway induced is organ dependent as Caspase 9 is induced in mid-gut but not in pronephros. These results indicate that MacroGard® feeding alone or in combination with other pathogenic factors did not induce significant apoptosis in immune organs.

Ligula intestinalis (Cestoda: Pseudophyllidea): an ideal fish-metazoan parasite model?

Since its use as a model to study metazoan parasite culture and in vitro development, the plerocercoid of the tapeworm, Ligula intestinalis, has served as a useful scientific tool to study a range of biological factors, particularly within its fish intermediate host. From the extensive long-term ecological studies on the interactions between the parasite and cyprinid hosts, to the recent advances made using molecular technology on parasite diversity and speciation, studies on the parasite have, over the last 60 years, led to significant advances in knowledge on host-parasite interactions. The parasite has served as a useful model to study pollution, immunology and parasite ecology and genetics, as well has being the archetypal endocrine disruptor.

Regionally acquired intestinal failure data suggest an underestimate in national service requirements.

UNLABELLED: OBJECTIVES, SETTING AND PATIENTS: With complete case referral for prolonged parenteral nutrition (PN) beyond term equivalent, serving a stable population of 1.25 million people, we describe the long-term outcome and survival of patients referred to an intestinal failure (IF) nutrition support team over the first 8 years of existence at a regional paediatric centre, and extrapolate to potential numbers of national home parenteral nutrition (HPN) cases and intestinal transplantation data. DESIGN AND OUTCOME MEASURES: Retrospective analysis detailing patient demographics, interventions, use of HPN, occurrence of intestinal failure-associated liver disease (IFALD), and outcomes of enteral adaptation, survival, and referral for and receipt of organ transplantation. RESULTS: 23 patients were referred over 8 years, 20 being PN dependent within the neonatal period. Diagnoses included short bowel syndrome (SBS) (18), neuromuscular abnormalities (4) and congenital enterocyte disorder (1). 12 696 days of PN were delivered with 314 confirmed episodes of sepsis at a median of 12 episodes per patient. 144 central venous catheters (CVCs) were required at a median of four per patient. IFALD occurred in 17 (73%) patients, with 10 (44%) referred for transplant assessment. Thirteen (56%) children received HPN. Overall mortality was 44%. A significant predictor for survival in the SBS group was residual bowel >40 cm (82% vs 28%, p = 0.049). CONCLUSIONS: Survival for IF at 56% was lower than reported from non-UK supra-regional centres, and nationally collected data, possibly reflecting pre-selected referral populations. Data from regional centres with complete ascertainment may be important both when counselling parents and when planning regional and national HPN and IF specialist services.

Characterisation of cDNAs of key genes involved in apoptosis in common carp (Cyprinus carpio L.).

Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Many of the cell deaths that occur during normal embryological development and during normal tissue turnover display the morphological hallmarks of apoptosis. In the last 20 or so years a better biochemical picture of how the process occurs has been produced, at least in higher vertebrates. It is now widely accepted that many of the proteolytic cleavages occurring during apoptosis are mediated by caspases, the activation of which, in turn, has been found to be tightly regulated. The current study focuses on the sequencing and analysis of key genes involved in the apoptotic process, based on sequence similarity to known apoptosis genes from genetic models such as zebrafish (Danio rerio) and other vertebrates. The present study identifies key components of the apoptotic process in common carp (Cyprinus carpio L.), which in turn can be used, for example, to monitor the fate of the cellular components of the immune system after an immune challenge.

Pollutant-induced effects on immunological and physiological interactions in aquatic host-trematode systems: implications for parasite transmission.

Under conditions of pollution both host and parasite are susceptible to the pathogenic effects of toxicants, which in turn may result in detrimental changes to their immunological and physiological processes. Digenetic trematodes, which encompass species of both medical and economic importance, possess complex life cycles and are common parasites of both vertebrates and molluscs. The combined stress induced by pollution and parasitism influences the physiology of the host which can have implications not only on host survival but also on the functional biology of resident parasite populations. The present paper reviews the effects of pollutants on the immunology and physiology in both vertebrate and molluscan host-trematode systems and the implications for parasite transmission.

The tapeworm Ligula intestinalis (Cestoda: Pseudophyllidea) inhibits LH expression and puberty in its teleost host, Rutilus rutilus.

The tapeworm Ligula intestinalis occurs in the body cavity of its cyprinid second intermediate host, in this study the roach Rutilus rutilus, and inhibits host gonadal development. The mechanism by which infected fish are prevented from reproducing is unknown. Comparison of parameters, such as body length and weight, and condition factor and age, between infected and uninfected individuals, indicated only minor effects of parasitism on growth and condition. In contrast, seasonal gonadal development, as observed in uninfected fish, did not occur in infected fish, and gonads remained small and blocked at the primary oocyte stage in female roach. As immature ovaries and testes are still present, the parasite is presumed to act upon the brain-pituitary-gonadal axis of the fish to inhibit further development of reproductive organs. We investigated the Ligula/fish interaction at the level of the pituitary gland by determination of gonadotrophin (LH) content using a heterologous RIA for carp (Cyprinus carpio) LHbeta subunit. The results indicated that the pituitary glands of infected roach contained approximately 50% less LH than non-infected fish. After the cloning and sequencing of roach LHbeta subunit, we measured roach LHbeta mRNA levels by real-time RT-PCR. A corresponding 50% reduction in LHbeta mRNA pituitary levels was determined. These results reflect a significant and measurable effect of parasitism on the pituitary gland, and lend support to the hypothesis that excretory/secretory products released from the parasite interact with the brain-pituitary-gonadal axis of the fish host and thus inhibit gonadal development.

Interaction between the blood fluke, Sanguinicola inermis and humoral components of the immune response of carp, Cyprinus carpio.

The effect of Sanguinicola inermis on serum antibody and complement activity in Cyprinus carpio was assessed using an ELISA and haemolytic assays. Possible immune evasion strategies were assessed using immunodetection of host proteins on the surface of the parasite. Carp acclimatized to 20 or 25 degrees C were infected by exposure to 500 cercariae or injected intraperitoneally with 150 cercariae, and serum monitored over a 63-day period. In cercariae-injected carp, irrespective of time and temperature, a significant increase occurred in complement activity being greatest at 25 degrees C. In addition, fish exposed to the cercariae of S. inermis and maintained at 20 degrees C the level of complement activity was significantly higher after 5 weeks compared to controls. At 20 degrees C intraperitoneal injections of parasites increased serum antibody levels which peaked after 7 days. In contrast, at 25 degrees C, antibody levels were maintained over 63 days. Exposure of fish to infection did not appear to stimulate antibody production. Immunofluorescence studies revealed 'host-like' molecules on the surface of the cercarial body exposed to carp serum and adult flukes obtained directly from the fish or cultured for 24 h in L15 medium. The possible role of 'host-like' molecules in immune evasion is discussed and the response at different temperatures is related to infection dynamics.

Inflammatory interactions in fish exposed to pollutants and parasites: a role for apoptosis and C reactive protein.

Although previous studies have highlighted the inflammatory responses of fish infected with parasites and exposed to pollutants, very little is known about how these two stressors interact within the fish. In this review, which also contains original data, the effect of these two parameters on the fish inflammatory response is assessed and, in particular, the role of apoptosis and the acute phase protein, C reactive protein, is evaluated. In Cyprinus carpio exposed to 0.5 mg NH4+ l(-1) or 0.1 mg Cd2+ l(-1) and experimentally infected with the blood fluke, Sanguinicola inermis, the pollutant type and the order in which the fish experiences the parasite and toxicant, significantly affects the ultrastructural appearance and cellular content of the pronephros and thymus. This is reflected in the intensity of infection where the pollutant appears to have less effect on an established infection. Both stressors, pollutant and infection, may mediate their effects via the endocrine system. Studies have revealed that cortisol at 100 ng ml(-1) is able to induce apoptosis in pronephric cells of carp and that an increase in apoptosis is associated with an increase in phagocytosis in this immune organ. In addition, C reactive protein, which is used as a biomarker of the inflammatory response in humans and other mammals, is evaluated as a possible indicator of physiological states in fish exposed to pathogens and pollutants.

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.

Ammonia-induced cellular and immunological changes in juvenile Cyprinus carpio infected with the blood fluke Sanguinicola inermis.

Immunological and structural changes in the thymus and pronephros of Cyprinus carpio infected with the blood fluke, Sanguinicola inermis for 30 days, and exposed to 0.5 mg NH4+/1 for 48 and 168 h were investigated. Ultrastructural observations revealed cell disruption and highly vacuolated cytoplasm in the thymus. Of the cells that remained intact there was a significant increase in thrombocytes after 48 h exposure to the pollutant. In addition, there was a decrease in lymphocytes following exposure to ammonia at both time-periods studied. In contrast the pronephros of fish exposed to the pollutant underwent relatively mild changes in cellular architecture although ammonia and time of exposure had significant effects on the proportions of several leucocyte types. A significant decrease in neutrophils, thrombocytes and lymphocytes occurred in fish exposed to the pollutant for 168 h. Pronephric lymphocyte stimulation (cpm) by Con A and PWM increased in vitro, whereas the stimulation index was reduced in infected fish exposed to ammonia. Changes in the immune organs of S. inermis-infected carp treated with pollutant were both organ- and time-specific. The possible reasons for this are discussed and significance in relationship to parasitization assessed.

Effects of Bothriocephalus acheilognathi on the polarization response of pronephric leucocytes of carp, Cyprinus carpio.

An in vitro assay was used to examine the effect of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda: Pseudophyllidea) on the polarization response of pronephric leucocytes of carp, Cyprinus carpio. Leucocytes, isolated from naive, naturally-infected fish and carp injected intraperitoneally with cestode extracts, were exposed to parasite extracts (protein concentrations 0-10.0 microg ml-1), for up to 24 h in the presence or absence of carp serum. In general, polarization responses of the pronephric leucocytes, primarily neutrophils and eosinophils, increased with incubation time although there was no significant difference in the response induced by the different protein concentrations. Differences in the polarization response were, however, observed in naive, naturally infected and injected fish and the cells responded differently in the presence and absence of carp serum. In the absence of carp serum the polarization response of pronephric leucocytes in vitro was significantly reduced with cells obtained from injected and naturally infected fish compared with those obtained from naive carp. This suppression of leucocyte migration was however reduced by the addition of carp serum to the in vitro system. The role of this interaction between the possible suppression of polarization induced by the parasite and stimulation by serum is discussed.

Antibody response of carp, Cyprinus carpio to the cestode, Bothriocephalus acheilognathi.

The humoral antibody response and the number of pronephric antibody-secreting cells were examined in naturally Bothriocephalus acheilognathi-infected carp. Cyprinus carpio, and in those injected intraperitoneally with an extract of the cestode. In the extract-injected fish, specific antibody was detected 3 weeks after a second injection given 2 weeks after the primary injection, and antibody levels persisted for more than 200 days. A third injection also enhanced the antibody level in the extract-injected carp. The numbers of antibody-secreting cells were significantly higher in carp injected 3 times with the extract than in the control. In naturally-infected fish, the serum antibody levels and the number of pronephric antibody-secreting cells were higher in infected fish than in uninfected individuals although this difference was not statistically significant. The relevance of these results to immune protection against infection is discussed.

Interactions between rat C-reactive protein and adult Hymenolepis diminuta.

Interactions between adult Hymenolepis diminuta and rat C-reactive protein (CRP) were investigated in vivo and in vitro. Using an ELISA technique, serum levels of CRP were monitored in rats infected with 100 cysticercoids. Although infection increased the level of this protein in the early stages of parasitization, the increase was not significant until 35 days post-infection (p.i.). Secondary infections did not enhance the response. When H. diminuta was cultured in the presence of CRP, reduced worm motility and opaque areas were observed and electron microscopical studies revealed shedding of microtriches and lysis of the tegument. Initially, damage was restricted to the strobila which correlated with the regional distribution of phosphorylcholine as visualized using immunofluorescence.

The in vitro effect of Khawia sinensis on leucocyte activity in carp (Cyprinus carpio).

The interactions between Khawia sinensis (Cestoda: Caryophyllidea), a pathogenic tapeworm of carp, and host leucocytes has been investigated in vitro by monitoring blastogenesis in pronephric, splenic and thymic lymphocytes and apoptosis of pronephric leucocytes. Both parasite homogenate and excretory/secretory (E/S) products were found to suppress lymphocyte blastogenesis at high concentrations (346.6-98.8 microg ml-1) whilst cell stimulation occurred at low concentrations (49.4-19.8 microg ml-1). This differential affect on leucocyte activity may not be associated with apoptosis in vitro as the parasite E/S products did not significantly affect programmed cell death as monitored by acridine orange and DNA analysis. The parasite effect on leucocyte activity is discussed and its relationship to host susceptibility is evaluated.

The effects of pollutants on the immune response of fish: implications for helminth parasites.

The effects of pollution on the immune response of fish are important in the dynamics of host/parasite interactions. Although there has been no definitive study that correlates a pollutant with the change in immune status of the fish and subsequent susceptibility to parasitic infection, there have been investigations which have noted changes in several immune parameters. The variable effects of pollutants on innate immunity, antibody levels and leucocyte numbers, migration and activity are reviewed and the consequences of observed changes for parasite fauna assessed. Studies on the exposure of C. carpio to cadmium have revealed an increase in apoptosis in pronephric cells. The implication of this immunosuppression is discussed.

Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae).

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.