• Keine Ergebnisse gefunden

7.5 Conclusions

In the present study, the Ussing chamber technique was adapted to describe electrogenic activities of isolated carp intestine, especially a phlorizin sensitive electrogenic cotransporter activity that can be only explained by SGLT1. This activity mainly took part in the mid intestine of carp, in particular in lower mid intestine. Partial sequences of carp SGLT1, claudin-1, -2, -3b, -3c, -7, -11, -23, and -30 were isolated, their tissue expression profiles were described, and their expression levels along the gut axis were observed on the transcription level. SGLT1 is a highly conserved gene and carp SGLT1 is highly similar to the gene of zebrafish. Compared to its expression in other tissues, the mRNA expression of carp SGLT1 was high in intestine and kidney. In gut it was higher especially in the upper mid intestine. Carp claudins showed a tissue-specific expression pattern and in the intestine most of the genes were highly expressed in posterior part of gut.

Intestinal levels of expression of carp SGLT1 and claudin genes were affected by challenges with pathogens and during a feeding experiment. In a systemic infection with KHV intestinal expression of carp SGLT1 mRNA was decreased and transcriptional levels of claudin-2, -3c, -11, and -23 were increased. Alterations in the expression of claudin genes were also observed after a feed modulation and a challenge of carp with Aeromonas hydrophila. A change from normal feed to experimental feeds increased mRNA levels of claudin-3c, -7, -23, and 30 in the posterior intestine and reduced the transcriptional level of claudin-2 in middle intestine. Supplementation of -glucan to the experimental diet did not influence mRNA expression of SGLT1 or claudin genes. However a fortification of the diet with this polysaccharide which is composed of D-glucose monomers and which was derived from Saccharomyces cerevicae cell walls was associated with differential responses of claudins to an administration of Aeromonas hydrophila into the gut. In fish fed without -glucan supplementation, an intubation of the bacterium elevated the levels of claudin-11 and -30 mRNA while in carp which received the -glucan enriched diet, the challenge with the bacterium raised mRNA levels of claudin-3c and -23. Additionally, feeding with -glucan seemed to protect carp from inflammation processes associated with the bacterial intubation.

9 )

Summary

Hamdan Syakuri

Studies of Intestinal Barrier Functions of Common Carp, Cyprinus carpio, under Feeding Modulation and Pathogen Challenge

The epithelium which covers the mucosal surface of the intestine of animals has important barrier functions which include the selective uptake of nutrients, the traffic of ions and water, and the protection of intestinal tissues as well as tissue beneath the mucosa from luminal antigens, toxins, or pathogenic invaders. For an improvement of fish production and fish health in aquaculture, more comprehensive knowledge on the diverse functions of the intestine in fish is needed. In aquaculture, supplies with highly digestable, supportive feed on a commercial basis and the prevention/ treatment of outbreaks of infectious diseases are issues which mainly challenge the production of fish including common carp for along period. The present work therefore was aimed to investigate the transporter protein SGLT1 and inter membrane proteins from the claudin family as two important elements of the intestinal barrier which are involved in selective permeability and the protective function of the intestine.

The Ussing chamber technique was implemented to describe electrogenic activities across the isolated carp intestine, in particular SGLT1 activity. PCR-based molecular assays were used to identify carp SGLT1 and claudin genes, describe their transcriptional profiles, and evaluate the modulation of their expression during the challenge with pathogens or the change of feeding. The potential dietary immunomodulant -glucan and two important disease causaing agents in carp aquaculture, koi herpesvirus (KHV) and Aeromonas hydrophila were used as experimental models.

In carp intestinal glucose uptake that is mediated by SGLT1 exclusively takes place in the middle intestine, especially in the lower mid gut. Because it is known as a highly conserved gene, carp SGLT1 is very similar to the gene from other piscine

9 )

species, in particular from zebrafish. The mRNA of this gene was highly expressed in the intestine, in particular in the upper mid intestine, in kidney, and at lower levels it was expressed in other tissues such as brain, liver, and gills. Intestinal transcription of the carp SGLT1 gene was reduced under KHV infection and it was not altered during a challenge of carp gut with Aeromonas hydrophila and after the modulation of feeding. Partial DNA fragments of eight carp claudin genes were isolated. Some of the claudin genes of carp such as claudin-7 and -11 seemed to be expressed in various tissues while some others like claudin-2 and -23 showed more tissue-specific expression profiles. In carp intestine, an infection with koi herpesvirus (KHV) increased mRNA expression of some claudin encoding genes such as claudin2, 3, -11, and -23. A change of the experimental feeding increased transcriptional levels of claudin-3c, -7, -23, and 30 in the posterior intestine and reduced the expression of claudin-2 mRNA in the middle intestine. However feeding of carp with -glucan did not influence mRNA expression of the genes encoding tight junction proteins, it appeared to prevent an intestinal inflammation and to differentiate the transcriptional response of claudin genes due to a bacterial challenge. Shortly after intubation of the bacterium carp that received a diet without -glucan showed an upregulation of claudin-11 and -30 transcriptions while carp which were fed with a -glucan containing diet indicated an upregulation of claudin-3c and -23.

/

Zusammenfassung

Hamdan Syakuri

Studien zur intestinalen Barriere von Karpfen (Cyprinus carpio) nach Futterumstellung und unter Infektion mit Pathogenen

Das Epithel, das die mukosale Oberfläche im Darm von Tieren auskleidet, erfüllt wichtige Barrierefunktionen, die die selektive Aufnahme von Nährstoffen und den Transport von Ionen und Wasser ebenso einschließt wie den Schutz der Mukosa und der Gewebe unter der Mukosa vor luminalen Antigenen, Toxinen oder vor pathogenen Eindringlingen. Zur Verbesserung von Fischproduktion und Fischgesundheit in der Aquakultur ist eine umfangreichere Kenntnis über diese unterschiedlichen Funktionen im Darm von Fischen notwendig. In der Aquakultur sind insbesondere die Versorgung der Fische mit hoch verdaulichem, gehaltvollem und preiswertem Futter, der Schutz der Fische vor Infektionserkrankungen bzw. die Behandlung von Infektionserkrankungen Herausforderungen, die die Produktion von Fischen einschließlich des Speisekarpfens seit langem erheblich belasten. Die vorliegende Arbeit untersuchte deshalb den Kotransporter SGLT1 und Intermembran-Proteine aus der Claudin-Familie als zwei wichtige Elemente der intestinalen Barriere, die an der selektiven Permeabilität sowie der Schutzfunktion des Darms beteiligt sind.

Untersuchungen in Ussingkammern wurden zur Beschreibung der elektrogenen Vorgänge an isoliertem Darmgewebe von Karpfen, insbesondere der Aktivität von SGLT1 etabliert. Außerdem wurden molekulargenetische, auf PCR basierende Untersuchungen ausgeführt, um das SGLT1 codierende Gen und Claudin codierende Gene zu identifizieren, das Transkriptionsmuster dieser Gene in Karpfengeweben zu beschreiben, sowie um die Modulation der Expression dieser Gene unter Infektion von Karpfen mit Pathogenen oder nach einer Futterumstellung zu untersuchen. Als Versuchsmodelle wurden die Verfütterung des Immunmodulators Beta-Glukan als Futterzusatz, sowie die beiden in der Aquakultur

/

von Karpfen bedeutsamen Pathogene Koi Herpesvirus (KHV) und Aeromonas hydrophila verwendet.

Der durch SGLT1 vermittelte Glukosetransport erfolgt bei Karpfen im Mitteldarm, insbesondere im posterioren Abschnitt des Mitteldarms. Weil das SGLT1 codierende Gen als hoch konserviert bekannt ist, weist die Nucleotidsequenz des SGLT1 codierend Gens von Karpfen weist eine hohe Übereinstimmung mit der Nukleotidsequenz des Gens von anderen Fischarten und insbesondere vom Zebrafisch auf. Die mRNA dieses Gens war im Darm, und hier vor allem im vorderen Mitteldarm hoch exprimiert, ebenso wie in der Niere; in geringerem Maße war es in anderen Organen, wie Gehirn, Leber und Kiemen exprimiert. Die Expression dieses Gens war bei Karpfen unter Infektion mit KHV verringert, sie war aber nach einer oralen Verabreichung von Aeromonas hydrophila oder nach einer Futterumstellung nicht verändert. Von acht Claudin codierenden Genen wurden Genfragmente isoliert.

Einige dieser Claudin-Genen, wie Claudin-7 und -11 waren in unterschiedlichen Geweben von Karpfen exprimiert, während andere Gene, wie Claudin-2 oder -23 mehr gewebespezifische Expressionsprofile aufwiesen. Bei Karpfen Darm, hatte eine Infektion mit Herpesvirus koi (KHV) eine Erhöhung der mRNA Expression einiger Claudin kodierenden Gene wie Claudin-2, -3, -11, und -23. Eine Umstellung des im Experiment verabreichten Futters hatte eine Erhöhung der Transkriptonsraten von Claudin 3c, -7, -23 und -30 im Enddarm zur Folge und eine Verringerung der Expression von Claudin-2 mRNA im Mitteldarm. Das Verfüttern von Beta-Glukanen beeinflusste die mRNA –Expression der Gene, die für die untersuchten Transmembranproteine kodieren, jedoch nicht. Allerdings schien die Verfütterung von Beta-Glukanen bei Karpfen nach oraler Verabreichung von Aeromonas hydrophila das Auftreten von Entzündungsreaktionen zu verhindern und eine Expression von Claudin-Genen zu beeinflussen. Kurze Zeit nach der Verabreichung der Bakterien war im Darm von Karpfen, die mit einer Diät ohne Beta Glukanzusatz gefüttert wurden, eine erhöhte Transkription von Claudin-11 und -30 zu erkennen, während im Darm von Karpfen, die eine Diät mit Beta-Glukan Zusatz erhielten, Claudin 3c und -23 hochreguliert waren.

8

References

ABBOTT, S. L., W. K. CHEUNG and J. M. JANDA (2003):

The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes.

J Clin Microbiol 41, 2348-2357

ADAMEK, M., K. L. RAKUS, J. CHYB, G. BROGDEN, A. HUEBNER, I. IRNAZAROW and D. STEINHAGEN (2012):

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

Fish & Shellfish Immunology 33, 482-493 AHMED, M. and M. H. LORICA (2002):

Improving developing country food security through aquaculture development-lessons from Asia.

Food Policy 27, 125-141

ALEXANDRE, M. D., B. G. JEANSONNE, R. H. RENEGAR, R. TATUM and Y.-H.

CHEN (2007):

The first extracellular domain of claudin-7 affects paracellular Cl permeability.

Biochemical and Biophysical Research Communications 357, 87-91

ALTHOFF, T., H. HENTSCHEL, J. LUIG, H. SCHÜTZ, M. KASCH and R. K. H.

KINNE (2007):

Na+-D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 292, R2391-R2399

ALTSCHUL, S. F., W. GISH, W. MILLER, E. W. MYERS and D. J. LIPMAN (1990):

Basic local alignment search tool.

J Mol Biol 215, 403-410

ALVARADO, F. and R. K. CRANE (1962):

Phlorizin as a competitive inhibitor of the active transport of sugars by hamster small intestine, in vitro.

Biochimica et Biophysica Acta 56, 170-172

AMASHEH, S., N. MEIRI, A. H. GITTER, T. SCHÖNEBERG, J. MANKERTZ, J. R. D.

SCHULZKE and M. FROMM (2002):

Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.

Journal of Cell Science 115, 4969-4976

8

AMASHEH, S., T. SCHMIDT, M. MAHN, P. FLORIAN, J. MANKERTZ, S. TAVALALI, A. H. GITTER, J.-D. SCHULZKE and M. FROMM (2005):

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.

Cell and Tissue Research 321, 89-96

AMASHEH, M., S. SCHLICHTER, S. AMASHEH, J. MANKERTZ, M. ZEITZ, M.

FROMM and J. R. D. SCHULZKE (2008):

Quercetin Enhances Epithelial Barrier Function and Increases Claudin-4 Expression in Caco-2 Cells.

The Journal of Nutrition 138, 1067-1073

AMASHEH, S., M. FROMM and D. GÜNZEL (2011):

Claudins of intestine and nephron – a correlation of molecular tight junction structure and barrier function.

Acta Physiologica 201, 133-140

AMAT, C., J. M. PLANAS and M. MORETO (1996):

Kinetics of hexose uptake by the small and large intestine of the chicken.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 271, R1085-R1089

ANDRAS, I. E. and M. TOBOREK (2011):

HIV-1-induced alterations of claudin-5 expression at the blood-brain barrier level.

Methods Mol Biol 762, 355-370

ANGELOW, S., R. AHLSTROM and A. S. YU (2008):

Biology of claudins.

Am J Physiol Renal Physiol 295, F867-876 ANGKA, S. L., T. J. LAM and Y. M. SIN (1995):

Some virulence characteristics of Aeromonas hydrophila in walking catfish (Clarias gariepinus).

Aquaculture 130, 103-112

ANISIMOVA, M. and O. GASCUEL (2006):

Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative.

Syst Biol 55, 539-552 AOKI, T. (1988):

Drug-resistant plasmids from fish pathogens.

Microbiol Sci 5, 219-223

AOKI, T. and A. TAKAHASHI (1987):

Class D tetracycline resistance determinants of R plasmids from the fish pathogens Aeromonas hydrophila, Edwardsiella tarda, and Pasteurella piscicida.

Antimicrob Agents Chemother 31, 1278-1280

8

ARDÓ, L., Z. JENEY, A. ADAMS and G. JENEY (2010):

Immune responses of resistant and sensitive common carp families following experimental challenge with Aeromonas hydrophila.

Fish & Shellfish Immunology 29, 111-116

ASCHENBACH, J., K. STEGLICH, G. GÄBEL and K. HONSCHA (2009):

Expression of mRNA for glucose transport proteins in jejunum, liver, kidney and skeletal muscle of pigs.

Journal of Physiology and Biochemistry 65, 251-266

BAGHERIE-LACHIDAN, M., S. I. WRIGHT and S. P. KELLY (2008):

Claudin-3 tight junction proteins in Tetraodon nigroviridis: cloning, tissue-specific expression, and a role in hydromineral balance.

American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 294, R1638-R1647

BAGNI, M., N. ROMANO, M. G. FINOIA, L. ABELLI, G. SCAPIGLIATI, P. G.

TISCAR, M. SARTI and G. MARINO (2005):

Short- and long-term effects of a dietary yeast -glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax).

Fish & Shellfish Immunology 18, 311-325

BAKKE, A. M., C. GLOVER and A. KROGDAHL (2011):

Feeding, Digestion and Absorption of Nutrients.

In: Fish Physiology, Volume 30, The Multifunctional gut of fish (M. Grosell, A.P.

Farrell, and C.J. Brauner, Editors) Academic Press, San Diego, S. 57-111 BALON, E. K. (1995):

Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers.

Aquaculture 129, 3-48

BANAN, A., J. Z. FIELDS, H. DECKER, Y. ZHANG and A. KESHAVARZIAN (2000):

Nitric oxide and its metabolites mediate ethanol-induced microtubule disruption and intestinal barrier dysfunction.

J Pharmacol Exp Ther 294, 997-1008

BANAN, A., J. Z. FIELDS, Y. ZHANG and A. KESHAVARZIAN (2001):

iNOS upregulation mediates oxidant-induced disruption of F-actin and barrier of intestinal monolayers.

Am J Physiol Gastrointest Liver Physiol 280, G1234-1246

BANERJEE, S. K., K. R. MCGAFFIN, N. R. M. PASTOR-SOLER and F. AHMAD (2009):

SGLT1 is a novel cardiac glucose transporter that is perturbed in disease states.

Cardiovascular Research 84, 111-118

8

BERCOVIER, H., Y. FISHMAN, R. NAHARY, S. SINAI, A. ZLOTKIN, M. EYNGOR, O. GILAD, A. ELDAR and R. HEDRICK (2005):

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BMC Microbiology 5, 13

BERKES, J., V. K. VISWANATHAN, S. D. SAVKOVIC and G. HECHT (2003):

Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.

Gut 52, 439-451

BIHLER, I. and R. K. CRANE (1962):

Studies on the mechanism of intestinal absorption of sugars V. The influence of several cations and anions on the active transport of sugars, in vitro, by various preparations of hamster small intestine.

Biochimica et Biophysica Acta 59, 78-93

BONDAD-REANTASO, M. G., R. P. SUBASINGHE, J. R. ARTHUR, K. OGAWA, S.

CHINABUT, R. ADLARD, Z. TAN and M. SHARIFF (2005):

Disease and health management in Asian aquaculture.

Veterinary Parasitology 132, 249-272

BRETZINGER, A., T. FISHER-SCHERL, M. OUMOUNA, R. HOFFMANN and U.

TRUYEN (1999):

Mass mortalities in koi carp, Cyprinus carpio, associated with gill and skin disease.

Bull. Eur. Ass. Fish Pathol. 19, 182-185

BREVES, G., B. SCHRÖDER and A. MUSCHER (2010):

Luminal and endocrine factors for regulation of intestinal monosaccharide and Ca2+

transport.

Livestock Science 134, 4-10

BROWN, G. D. and S. GORDON (2003):

Fungal beta-glucans and mammalian immunity.

Immunity 19, 311-315

BUCHMANN, K., H. C. SLOTVED and D. DANA (1995):

Gill parasites from Cyprinus carpio in Indonesia.

Aquaculture 129, 437-438

BUI, P., M. BAGHERIE-LACHIDAN and S. P. KELLY (2010):

Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

Molecular and Cellular Endocrinology 317, 120-126

8

BUSTIN, S. A. (2002):

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

J Mol Endocrinol 29, 23-39

CARVALHO-CASTRO, G. A., C. O. LOPES, C. A. G. LEAL, P. G. CARDOSO, R. C.

LEITE and H. C. P. FIGUEIREDO (2010):

Detection of type III secretion system genes in Aeromonas hydrophila and their relationship with virulence in Nile tilapia.

Veterinary Microbiology 144, 371-376

CASCON, A., J. YUGUEROS, A. TEMPRANO, M. SANCHEZ, C. HERNANZ, J. M.

LUENGO and G. NAHARRO (2000):

A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila.

Infect Immun 68, 3233-3241

CASNEUF, V. F., P. FONTEYNE, N. VAN DAMME, P. DEMETTER, P. PAUWELS, B. DE HEMPTINNE, M. DE VOS, C. VAN DE WIELE and M. PEETERS (2008):

Expression of SGLT1, Bcl-2 and p53 in Primary Pancreatic Cancer Related to Survival.

Cancer Investigation 26, 852-859

CHAKRABORTY, T., M. A. MONTENEGRO, S. C. SANYAL, R. HELMUTH, E.

BULLING and K. N. TIMMIS (1984):

Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotonic enterotoxin.

Infect Immun 46, 435-441

CHASIOTIS, H. and S. P. KELLY (2011):

Effect of cortisol on permeability and tight junction protein transcript abundance in primary cultured gill epithelia from stenohaline goldfish and euryhaline trout.

General and Comparative Endocrinology 172, 494-504 CHEN, L., D. M. SEGAL and D. C. MASH (1999):

Semi-quantitative reverse-transcriptase polymerase chain reaction: an approach for the measurement of target gene expression in human brain.

Brain Res Brain Res Protoc 4, 132-139

CHENG, L., C. Y. CHEN, M. A. TSAI, P. C. WANG, J. P. HSU, R. S. CHERN and S.

C. CHEN (2011):

Koi herpesvirus epizootic in cultured carp and koi, Cyprinus carpio L., in Taiwan.

J Fish Dis 34, 547-554

CHEVENET, F., C. BRUN, A. L. BANULS, B. JACQ and R. CHRISTEN (2006):

TreeDyn: towards dynamic graphics and annotations for analyses of trees.

BMC Bioinformatics 7, 439

8

CLARKE, L. L. (2009):

A guide to Ussing chamber studies of mouse intestine.

American Journal of Physiology - Gastrointestinal and Liver Physiology 296, G1151-G1166

CLELLAND, E. S., P. BUI, M. BAGHERIE-LACHIDAN and S. P. KELLY (2010):

Spatial and salinity-induced alterations in claudin-3 isoform mRNA along the gastrointestinal tract of the pufferfish Tetraodon nigroviridis.

Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 155, 154-163

COLEGIO, O. R., C. VAN ITALLIE, C. RAHNER and J. M. ANDERSON (2003):

Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture.

Am J Physiol Cell Physiol 284, C1346-1354

COLEGIO, O. R., C. M. VAN ITALLIE, H. J. MCCREA, C. RAHNER and J. M.

ANDERSON (2002):

Claudins create charge-selective channels in the paracellular pathway between epithelial cells.

Am J Physiol Cell Physiol 283, C142-147

COSTES, B., G. FOURNIER, B. MICHEL, C. DELFORGE, V. S. RAJ, B. DEWALS, L. GILLET, P. DRION, A. BODY, F. SCHYNTS, F. LIEFFRIG and A.

VANDERPLASSCHEN (2008):

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

J Virol 82, 4955-4964

COSTES, B., V. S. RAJ, B. MICHEL, G. FOURNIER, M. THIRION, L. GILLET, J.

MAST, F. LIEFFRIG, M. BREMONT and A. VANDERPLASSCHEN (2009):

The Major Portal of Entry of Koi Herpesvirus in Cyprinus carpio is the Skin.

J. Virol. 83, 2819-2830

CRANE, R. K. and P. MANDELSTAM (1960):

The active transport of sugars by various preparations of hamster intestine.

Biochimica et Biophysica Acta 45, 460-476

CUNNINGHAM, A. A., T. E. LANGTON, P. M. BENNETT, J. F. LEWIN, S. E.

DRURY, R. E. GOUGH and S. K. MACGREGOR (1996):

Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria).

Philos Trans R Soc Lond B Biol Sci 351, 1539-1557

8

DALMO, R. A. and J. BØGWALD (2008):

-glucans as conductors of immune symphonies.

Fish & Shellfish Immunology 25, 384-396 DASKALOV, H. (2006):

The importance of Aeromonas hydrophila in food safety.

Food Control 17, 474-483

DE FIGUEIREDO, J. and J. A. PLUMB (1977):

Virulence of different isolates of Aeromonas hydrophila in channel catfish.

Aquaculture 11, 349-354

DEREEPER, A., S. AUDIC, J. M. CLAVERIE and G. BLANC (2010):

BLAST-EXPLORER helps you building datasets for phylogenetic analysis.

BMC Evol Biol 10, 8

DEREEPER, A., V. GUIGNON, G. BLANC, S. AUDIC, S. BUFFET, F. CHEVENET, J. F. DUFAYARD, S. GUINDON, V. LEFORT, M. LESCOT, J. M. CLAVERIE and O.

GASCUEL (2008):

Phylogeny.fr: robust phylogenetic analysis for the non-specialist.

Nucleic Acids Res 36, W465-469

DILLER, I. C., Z. T. MANKOWSKI and M. E. FISHER (1963):

The effect of yeast polysaccharides on mouse tumors.

Cancer Res 23, 201-208

DISHON, A., A. PERELBERG, J. BISHARA-SHIEBAN, M. ILOUZE, M.

DAVIDOVICH, S. WERKER and M. KOTLER (2005):

Detection of carp interstitial nephritis and gill necrosis virus in fish droppings.

Appl Environ Microbiol 71, 7285-7291 DOGGETT, T. A. and J. E. HARRIS (1991):

Morphology of the gut associated lymphoid tissue of Oreochromis mossambicus and its role in antigen absorption.

Fish & Shellfish Immunology 1, 213-227 DOOLEY, J. S. and T. J. TRUST (1988):

Surface protein composition of Aeromonas hydrophila strains virulent for fish:

identification of a surface array protein.

Journal of Bacteriology 170, 499-506

DROZDOWSKI, L. A. and A. B. THOMSON (2006):

Intestinal sugar transport.

World J Gastroenterol 12, 1657-1670

8

EDGAR, R. C. (2004):

MUSCLE: multiple sequence alignment with high accuracy and high throughput.

Nucleic Acids Res 32, 1792-1797

EL-MATBOULI, M. and H. SOLIMAN (2011):

Transmission of Cyprinid herpesvirus-3 (CyHV-3) from goldfish to naive common carp by cohabitation.

Res Vet Sci 90, 536-539

EPPLE, H. J., J. MANKERTZ, R. IGNATIUS, O. LIESENFELD, M. FROMM, M.

ZEITZ, T. CHAKRABORTY and J. D. SCHULZKE (2004):

Aeromonas hydrophila Beta-Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells (HT-29/B6).

Infection and Immunity 72, 4848-4858

ESTERABADI, A. H., F. ENTESSAR and M. A. KHAN (1973):

Isolation and identification of Aeromonas hydrophila from an outbreak of haemorrhagic septicemia in snakes.

Can J Comp Med 37, 418-420

EVANS, M. J., T. VON HAHN, D. M. TSCHERNE, A. J. SYDER, M. PANIS, B.

WOLK, T. HATZIIOANNOU, J. A. MCKEATING, P. D. BIENIASZ and C. M. RICE (2007):

Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.

Nature 446, 801-805

EVELOFF, J., M. FIELD, R. KINNE and H. MURER (1980):

Sodium-cotransport systems in intestine and kidney of the winter flounder.

Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology 135, 175-182

FAO 2011.

Cultured Aquatic Species Information Programme Cyprinus carpio (Linnaeus, 1758) Food and Agriculture Organization of the United Nations.

FALCO, A., P. FROST, J. MIEST, N. PIONNIER, I. IRNAZAROW and D. HOOLE (2012):

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with beta-glucan supplements.

Fish & Shellfish Immunology 32, 1051-1057

FASANO, A., B. BAUDRY, D. W. PUMPLIN, S. S. WASSERMAN, B. D. TALL, J. M.

KETLEY and J. B. KAPER (1991):

Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.

Proceedings of the National Academy of Sciences 88, 5242-5246

8

FASANO, A., C. FIORENTINI, G. DONELLI, S. UZZAU, J. B. KAPER, K.

MARGARETTEN, X. DING, S. GUANDALINI, L. COMSTOCK and S. E. GOLDBLUM (1995):

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent