Hypoxemia-induced leptin secretion: a mechanism for the control of food intake in diseased fish.

Leptin is a potent anorexigen, but little is known about the physiological conditions under which this cytokine regulates food intake in fish. In this study, we characterized the relationships between food intake, O2-carrying capacity, liver leptin-A1 (lep-a1) gene expression, and plasma leptin-A1 in rainbow trout infected with a pathogenic hemoflagellate, Cryptobia salmositica. As lep gene expression is hypoxia-sensitive and Cryptobia-infected fish are anemic, we hypothesized that Cryptobia-induced anorexia is mediated by leptin. A 14-week time course experiment revealed that Cryptobia-infected fish experience a transient 75% reduction in food intake, a sharp initial drop in hematocrit and hemoglobin levels followed by a partial recovery, a transient 17-fold increase in lep-a1 gene expression, and a sustained increase in plasma leptin-A1 levels. In the hypothalamus, peak anorexia was associated with decreases in mRNA levels of neuropeptide Y (npy) and cocaine- and amphetamine-regulated transcript (cart), and increases in agouti-related protein (agrp) and pro-opiomelanocortin A2 (pomc). In contrast, in non-infected fish pair-fed to infected animals, lep-a1 gene expression and plasma levels did not differ from those of non-infected satiated fish. Pair-fed fish were also characterized by increases in hypothalamic npy and agrp, no changes in pomc-a2, and a reduction in cart mRNA expression. Finally, peak infection was characterized by a significant positive correlation between O2-carrying capacity and food intake. These findings show that hypoxemia, and not feed restriction, stimulates leptin-A1 secretion in Cryptobia-infected rainbow trout and suggest that leptin contributes to anorexia by inhibiting hypothalamic npy and stimulating pomc-a2.

Immunity against selected piscine flagellates.

This discussion is on immune response to Amyloodinium ocellatum, Cryptobia salmositica, Trypanoplasma borreli and Trypanosoma carassii. Piscidin and histone-like proteins enhance innate resistance to Amyloodinium. Fish that are naturally resistant to Cryptobia and Trypanoplasma can be bred. Cryptobia resistance in charr is controlled by a dominant Mendelian locus and protection is via the Alternative Pathway of Complement Activation. Studies on Cryptobia-tolerant charr may lead to production of transgenic Cryptobia-tolerant salmon. Innate response to T. borreli is associated with NO in macrophages. Transferrin regulates resistance and carp have been bred for transferrin genotypes. Recovered fish are protected from homologous challenge, and complement fixing antibodies are crucial in protection. Studies on antigens in T. carassii may lead to a vaccine. There are two vaccines against cryptobiosis; a single dose of the attenuated vaccine protects salmonids. On challenge fish inoculated with the metalloprotease-DNA vaccine do not have the disease and they recover faster.

Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica.

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50ngml(-1) of RU486 was more effective in inhibiting parasite replication in cultures with 7,000parasitesml(-1) than in cultures with 14,000parasitesml(-1). Also, 100ngml(-1) of RU486/ml was more effective than 50ngml(-1) in inhibiting parasite multiplication in the 14,000 parasitesml(-1) cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand-receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host-parasite interaction.

Duress without stress: Cryptobia infection results in HPI axis dysfunction in rainbow trout.

Despite clear physiological duress, rainbow trout (Oncorhynchus mykiss) infected with the pathogenic haemoflagellate Cryptobia salmositica do not appear to mount a cortisol stress response. Therefore, we hypothesized that the infection suppresses the stress response by inhibiting the key effectors of the hypothalamic-pituitary-interrenal (HPI) axis. To test this, we characterized the basal activity of the HPI axis and the cortisol response to air exposure in saline- and parasite-injected fish. All fish were sampled at 4 and 6 weeks post-injection (wpi). While both the treatment groups had resting plasma cortisol levels, the parasite-infected fish had lower levels of plasma ACTH than the control fish. Relative to the control fish, the infected fish had higher mRNA levels of brain pre-optic area corticotrophin-releasing factor (CRF) and pituitary CRF receptor type 1, no change in pituitary POMC-A1, -A2 and -B gene expression, higher and lower head kidney melanocortin 2 receptor mRNA levels at 4 and 6 wpi respectively and reduced gene expression of key proteins regulating interrenal steroidogenesis: StAR, cytochrome P450scc and 11ß-hydroxylase. The parasite-infected fish also had a reduced plasma cortisol response to a 60-s air exposure stressor. Superfusion of the head kidney tissues of the parasite-infected fish led to significantly lower ACTH-stimulated cortisol release rates than that observed in the control fish. These novel findings show that infection of rainbow trout with C. salmositica results in complex changes in the transcriptional activity of both central and peripheral regulators of the HPI axis and in a reduction in the interrenal capacity to synthesize cortisol.

Cortisol and dexamethasone increase the in vitro multiplication of the haemoflagellate, Cryptobia salmositica, possibly by interaction with a glucocorticoid receptor-like protein.

Cryptobia salmositica is a pathogenic haemoflagellate of Pacific salmon, Oncorhynchus spp., on the west coast of North America. The in vitro multiplication of the parasite was significantly enhanced by the addition of cortisol (within a range consistent with physiological levels in salmonid fishes; 10-50 ng ml(-1)) to the culture medium (MEM supplemented with FBS). However, higher cortisol concentrations (100 and 200 ng ml(-1)) either had no enhancing effects or resulted in lower replication rates compared with the controls. The synthetic glucocorticoid, dexamethasone (Dex), also stimulated the replication of the parasite and mifepristone (RU486), a synthetic steroid that has glucocorticoid receptor (GR) antagonist properties, inhibited the stimulatory actions of both cortisol and Dex, when added to the medium at a concentration of 100 ng ml(-1) co-culture with cortisol or Dex. Furthermore, the dose-dependent effects of glucocorticoids (cortisol and Dex) on the multiplication of the haemoflagellate were correlated with the initial size of the inocula. The study revealed a novel relationship between the parasite and its host, in which the host's cortisol is used by the parasite to enhance its replication. Also, since C. salmositica responds to both native and synthetic glucocorticoids and to the GR antagonist, RU486, and exhibits a biphasic (hormetic) response to the amount of cortisol in the medium, we propose that the glucocorticoid exerts its effects via an interaction with GR-like proteins in C. salmositica that are functionally similar to those present in vertebrate cells.

Small subunit ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 gene sequences of 21 strains of the parasitic scuticociliate Miamiensis avidus (Ciliophora, Scuticociliatia).

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes. Small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes were analyzed for 21 strains of M. avidus isolated from diseased olive flounder (Paralichthys olivaceus), ridged-eye flounder (Pleuronichthys cornutus), and spotted knifejaw (Oplegnathus fasciatus) in Korea and Japan (collected in 2003-2007). Analysis of SSU rRNA gene sequences (1,759 bp) indicates they are very conserved with less than 0.17% (3 nucleotides) differences suggesting that SSU rRNA are useful to identify M. avidus; however, the cox1 gene (900 bp) has higher variations with intraspecific divergences up to 5.67% (51 nucleotides). A distance tree of cox1 gene sequences based on a neighbor-joining analysis can separate 21 strains into five cox1 types (two heterogeneous clusters and three individual branches). The cox1-type matches with serotype of strains but do not reflect geographical origins, host species, or pathogenicity.

Immunological and therapeutic strategies against salmonid cryptobiosis.

Salmonid cryptobiosis is caused by the haemoflagellate, Cryptobia salmositica. Clinical signs of the disease in salmon (Oncorhynchus spp.) include exophthalmia, general oedema, abdominal distension with ascites, anaemia, and anorexia. The disease-causing factor is a metalloprotease and the monoclonal antibody (mAb-001) against it is therapeutic. MAb-001 does not fix complement but agglutinates the parasite. Some brook charr, Salvelinus fontinalis cannot be infected (Cryptobia-resistant); this resistance is controlled by a dominant Mendelian locus and is inherited. In Cryptobia-resistant charr the pathogen is lysed via the Alternative Pathway of Complement Activation. However, some charr can be infected and they have high parasitaemias with no disease (Cryptobia-tolerant). In infected Cryptobia-tolerant charr the metalloprotease is neutralized by a natural antiprotease, alpha2 macroglobulin. Two vaccines have been developed. A single dose of the attenuated vaccine protects 100% of salmonids (juveniles and adults) for at least 24 months. Complement fixing antibody production and cell-mediated response in vaccinated fish rise significantly after challenge. Fish injected with the DNA vaccine initially have slight anaemias but they recover and have agglutinating antibodies. On challenge, DNA-vaccinated fish have lower parasitaemias, delayed peak parasitaemias and faster recoveries. Isometamidium chloride is therapeutic against the pathogen and its effectiveness is increased after conjugation to antibodies.

Contribution of food deprivation to the immune response in rainbow trout (Oncorhynchus mykiss) vaccinated against Cryptobia salmositica and Aeromonas salmonicida.

The aims of the present study were to determine (a) the effectiveness of an attenuated live Cryptobia salmositica vaccine; (b) the effects of food deprivation on the immune response and its duration in rainbow trout (Oncorhynchus mykiss) immunised with a live C. salmositica vaccine or with a killed Aeromonas salmonicida vaccine. The fish were divided into three groups (I, II and III; 14 fish per group), those in Groups I and II were under food deprivation (0.40% of body weight), while Group III fish were fed to satiety. The study showed that the attenuated strain of C. salmositica did not cause anaemia and disease, and the fish were protected from clinical disease when they were challenged with virulent parasites. Parasitaemia in all fish vaccinated and challenged with virulent C. salmositica fluctuated and was relatively low; however, fish in Group III had higher parasitaemia than those in Groups I and II between weeks 8 and 14. The numbers of activated neutrophils increased [nitroblue tetrazolium (NBT) assay] after immunisation with both Cryptobia and Aeromonas vaccines and they remained high throughout the experiment. Antibody production (ELISA values) increased after vaccination and were slightly higher in Group III. ELISA titres against A. salmonicida increased after vaccination and decreased after 5 weeks. The titres increased again after the vaccinated fish were given booster, and they were higher than those in the first vaccinated fish.

Towards a metalloprotease-DNA vaccine against piscine cryptobiosis caused by Cryptobia salmositica.

Cysteine protease is a metabolic enzyme, whereas metalloprotease is the virulent factor in cryptobiosis caused by Cryptobia salmositica. Recombinant DNA vaccines were produced with the insertion of either the metalloprotease or cysteine protease gene of C. salmositica into plasmid vectors (pEGFP-N). As expected, fishes (Oncorhynchus mykiss and Salmo salar) injected intramuscularly with the metalloprotease-DNA (MP-DNA) vaccine (50 microg/fish) were consistently more anemic (lower packed cell volume, PCV) than controls (injected only with the plasmid) at 3-5 weeks post-inoculation. Also, there were no difference in PCV between fish injected with the cysteine-DNA plasmids and the controls. In addition, agglutinating antibodies against Cryptobia were detected only in the blood of MP-DNA-vaccinated fish at 5-7 weeks post-vaccination and not in cysteine-DNA plasmids and the control groups. MP-DNA-vaccinated fish when challenged with the pathogen had consistently lower parasitemia, delayed peak parasitemia, and faster recovery compared with the controls. All fish vaccinated with attenuated strain were protected when challenged with the pathogen; this positive control group confirmed that the two vaccines operate through different mechanisms.

Use of a live vaccine to modulate Cryptobia salmositica infections in Salmo salar.

The vaccine strain of Cryptobia salmositica multiplies in Atlantic salmon Salmo salar and it can modulate the severity of the disease in Cryptobia-infected individuals. Fish injected with the vaccine 3 d post-infection with C. salmositica had lower peak parasitaemias and higher antibody titres than infected fish given the vaccine 7 d post-infection or those infected fish that were not given the vaccine.

Susceptibility of sexually mature rainbow trout, Oncorhynchus mykiss to experimental cryptobiosis caused by Cryptobia salmositica.

Sexually mature rainbow trout, Oncorhynchus mykiss, were highly susceptible to cryptobiosis caused by Cryptobia salmositica. Spawning female trout were more susceptible (higher parasitaemia and mortality) than sexually mature males. Most infected female trout (seven of nine) with eggs died before or shortly after spawning; however, none of the nine infected sexually matured males or the uninfected fish died. There was no significant difference in the severity of the anaemia between infected male and female trout. All infected males developed exophthalmia, while this clinical sign was not seen in any of the infected females nor in uninfected trout. The addition of 17 beta-estradiol (at physiological level or higher) did not enhance in vitro multiplication of the Cryptobia; however, fresh plasma from sexually mature females or males when added to cultures significantly increased in vitro multiplication of the pathogen. In addition, plasma from sexually mature females were significantly better than those from males in promoting in vitro parasite multiplication. Parasite multiplication did not increase after plasma from sexually mature fish were heat inactivated.

A S-adenosylmethionine synthetase gene from the pathogenic piscine hemoflagellate, Cryptobia salmositica.

We report on the identification of a Cryptobia genomic DNA gene, predict it to encode a S-adenosylmethionine synthetase signature 1 motif and propose to name it S-adenosylmethionine synthetase (MAT). The open reading frame of MAT is 1,046 bp with 341 deduced amino acids. The MAT gene was identified using universal genome walking and Southern blot analysis revealed it to be a multi-copy gene. The S-adenosylmethionine synthetase of Cryptobia salmositica amino acid sequence is similar to those of other pathogenic kinetoplastids (Leishmania donovani 71%, Leishmania major 70%, Leishmania infantum 71%, Trypanosoma brucei 72%, Trypanosoma cruzi 70% and T. cruzi strain CL Brener 70%). The C. salmositica MAT has a conserved hexapeptide GAGDQG, which is widely found in bacteria, parasitic protozoans and also in humans. These suggest that MAT may have highly conserved functions such as regulation of gene expression and biosynthesis of a multitude of essential metabolites.

A cathepsin L-like cysteine proteinase gene from the protozoan parasite, Cryptobia salmositica.

The present study describes the identification of a cathepsin L-like cysteine proteinase gene (CYS) from the hemoflagellate Cryptobia salmositica. Genomic DNA sequence of cysteine proteinase was obtained by genome walking using degenerate primers. Specific primers were designed to amplify the cDNA of cysteine proteinase from mRNA by rapid amplification of cDNA ends-PCR. The open reading frame of CYS is 1,329 bp, with 443 deduced amino acids. Based on the sequence analysis, cysteine proteinase of C. salmositica is similar to the cathepsin L-like cysteine proteinase of kinetoplastid parasites such as Leishmania spp. and Trypanosoma spp. The identification of CYS proteinase gene could help to design cysteine proteinase specific inhibitors. Further studies are required to characterize the complete genomic organization of the cysteine proteinase.

A metalloproteinase gene from the pathogenic piscine hemoflagellate, Cryptobia salmositica.

We report the identification of a Cryptobia genomic DNA gene predicted to encode a hydrophobic protein containing a zinc metalloproteinase motif, HEXXH, and hence named it a major surface proteinase 1-like (MSP-1). The MSP-1 gene was identified using universal genome walking. Southern blot analysis revealed it to be a multicopy gene. Fragments of DNA encoding a segment homologous to the HEXXH motif of MSP-1 are widely found in bacteria, yeast, parasitic protozoans, plants, and animals including humans. These results suggest that the MSP-1 may have highly conserved functions, such as in intracellular proteolysis.

Innate cell-mediated immune response and peripheral leukocyte populations in Atlantic salmon, Salmo salar L., to a live Cryptobia salmositica vaccine.

The effects of a live Cryptobia salmositica (Kinetoplastida) vaccine on the humoral and cellular immune response, and changes in the peripheral leukocyte populations of Salmo salar were investigated. The vaccine produced detectable parasitemia in the blood which peaked at 5 weeks post-vaccination (w.p.v). Antibodies were detectable at 4 w.p.v. and the antibody titer increased as parasitemia declined. Respiratory burst activity in vaccinated fish was significantly higher than in control fish; the highest activity occurred with rising parasitemia and lower activity with declining parasitemia. There was a significant increase in the proportion of granulocytes (to total leukocytes) at 4 w.p.v. At 6 w.p.v., the proportion of lymphocytes and monocytes increased significantly and remained elevated. These results demonstrate innate (respiratory burst activity and an increase in the proportion of granulocytes corresponding to rising parasitemia) and adaptive (antibody production and increases in the proportion of monocytes and lymphocytes corresponding to declining parasitemia) immune responses to the live vaccine.

Experimental infections of Atlantic salmon Salmo salar with Spironucleus barkhanus.

Atlantic salmon Salmo salar L. (Salmonidae) were experimentally infected with Spironucleus barkhanus (Diplomonadida: Hexamitidae). Parasites were found in the blood 1 to 8 wk after infection, after which they disappeared from the blood and were found mainly in the internal organs (e.g. spleen and liver), eye socket or muscles. Mortality (38 out of 40 infected fish) occurred when fish had lesions in internal organs and/or on the body surface. Uninfected fish cohabiting with infected fish became infected after 4 wk, indicating direct transmission. There was no difference in susceptibility to spironucleosis between 3 different families of Atlantic salmon. All families developed the disease with a similar pattern of parasitaemia in the blood, similar clinical signs and gross pathology, and with very high mortality (29 out of 30). Clinical signs of systemic spironucleosis may include anemia, skin blisters, muscle ulcerations or unilateral exophthalmia. Gross pathologies include hemorrhaging of internal organs, splenomegaly or deformed (globulated) spleen, or granulomatous lesions in the spleen and liver.

Detection and quantification of Spironucleus barkhanus in experimentally infected Atlantic salmon Salmo salar.

The course of Spironucleus barkhanus (Diplomonadida: Hexamitidae) infection in Atlantic salmon Salmo salar L. (Salmonidae) has 2 distinct phases, a blood phase and a tissue phase. To detect and quantify an infection, 3 parasitological techniques, namely Wet Mount Examination (WME), Hematocrit Centrifuge Technique (HCT) and the Hemocytometer (HCM) were used. In addition, 1 immunological technique, enzyme-linked immunosorbent assay (ELISA), was developed to detect specific antibodies against S. barkhanus. This technique would be particularly useful for epidemiological studies where large numbers of fish had to be examined. It would also be a good technique to detect infection during the tissue phase of the disease when there were no or a low number of parasites in the blood.

Effect of Cryptobia salmositica-induced anorexia on feeding behavior and immune response in juvenile rainbow trout Oncorhynchus mykiss.

At 10 degrees C, rainbow trout Oncorhynchus mykiss (n = 13 per group) infected with Cryptobia salmositica Katz, 1951 became anorexic at 3 wk post-infection (w.p.i.), with feed-intake decreasing significantly from 1.33 to 0.94% body weight (b.w.). Anorexia was most severe at 4 w.p.i. (0.80% b.w.), coinciding with peak parasitemia (9.2 x 10(6) parasites ml blood(-1)) and anemia. At 8 w.p.i., fish had recovered their appetite although they still had contained detectable parasites (6.8 x 10(5) parasites ml(-1)) and were anemic (pack cell volume, PCV, of 24.4%). However at 5 degrees C, anorexia occurred at 5 w.p.i. (0.81% b.w.), and was most severe at 7 w.p.i. (0.40% b.w.). At 8 w.p.i. (0.43% b.w.), fish displayed high parasitemia (4.6 x 10(6) parasites ml(-1)) and low PCV (10.8%). Fish at 5 degrees C had lower gastric evacuation (GE) rates (GE48h) than 10 degrees C fish, however there were no differences between infected and naive fish at both temperatures. Before anorexia, there was no significant correlation between mean share of meal (MSM, a measure of how food was partitioned within a group) and coefficient of variation in feeding but this became significant during anorexia (p = 0.02 and p = 0.0002 at 10 and 5 degrees C respectively). Significant correlations were detected between b.w. and MSM before onset of anorexia at 10 degrees C (p = 0.005) and 5 degrees C (p = 0.02); this was maintained at 10 degrees C (p = 0.001) but not at 5 degrees C (p = 0.98). Fish on an anorexic diet (0.93% b.w.) responded well at 10 degrees C to a live C. salmositica vaccine; this could partly be due to constant antigenic stimulation by the live vaccine.

Genetic relationships among some species groups within the genus Lutzomyia (Diptera: Psychodidae).

Molecular data on Lutzomyia are very scarce, despite the fact that this genus includes all the species involved in the transmission of leishmaniasis in America. We examine the genetic relationships among eight morphologic groups within the Lutzomyia genus and two Brumptomyia species, using nine enzyme loci and the last 285 basepairs of the mitochondrial cytochrome b gene. The structure of the genetic variation among the species analyzed indicated a closer genetic relationship among members of a morphologic group than between members of different groups. The lower levels of variation recorded among these groups compared with that between Brumptomyia and Lutzomyia suggest a subgeneric status for all of these groups, including Psychodopygus. A maximum likelihood tree for the allozyme data and a neighbor-joining consensus tree for the mitochondrial DNA sequences showed a general agreement with morphologic groups, with only minor differences. Nyssomyia, Verrucarum and Micropygomyia formed separate monophyletic groups. Lutzomyia could not be separated from Psathyromyia, and both Migonei species, L. dubitans and L. migonei, grouped in different clades according to the host species they are found on.