Bacterial translocation and pathogenesis in Bacterial translocation and pathogenesis in the digestive tract of larvae and fry: What the digestive tract of larvae and fry: What are the indigenous bacteria and pathogens are the indigenous bacteria and pathogens

doing?doing?

E. Ringø1,2, R. Myklebust3, T.M. Mayhew4 and R.E. Olsen2

1 Aquaculture Protein Centre. Norwegian School of Veterinary Science, Tromsø, Norway.

2 Institute of Marine Research, Bergen, Norway.3 University of Bergen, Norway.4 University of Nottingham, UK.

Schematic drawing of the digestive tract of spotted wolffish Schematic drawing of the digestive tract of spotted wolffish ((Anarhichas minorAnarhichas minor Olafsen) fry; [1] oesophagus, [2] stomach, [3] Olafsen) fry; [1] oesophagus, [2] stomach, [3]

foregut, [4] midgut, and [5] hindgut.foregut, [4] midgut, and [5] hindgut.

The three major routes of The three major routes of infection in fish are through;infection in fish are through;

- skin- gills- gastrointestinal [GI]tract

When discussing whether the gastro-intestinal tract is a port of entry for pathogenic bacteria in fish. A important question arise: Are there any information Are there any information about bacterial translocation and about bacterial translocation and pathogenesis in the GI tract of larvae pathogenesis in the GI tract of larvae and fry?and fry?

TRANSLOCATION OF TRANSLOCATION OF BACTERIABACTERIA

Under normal conditions the intestinal mucosa functions as a barrier, preventing bacteria from escaping the intestinal lumen. Different critical conditions, however, may lead to loss of the barrier function, enabling bacteria and endotoxin to infect otherwise sterile tissues, a process termed translocationtranslocation.

INDIGENOUS BACTERIAINDIGENOUS BACTERIAThe intestinal microbiota in fish as in mammals are classified as autochthonousautochthonous(indigenousindigenous) or allochthonousallochthonous bacteria. Autochthonous bacteria are able to colonise and adhere to intestinal mucus or areas between the microvilli of the enterocytes.

Are the indigenous gut bacteria in larvae Are the indigenous gut bacteria in larvae and fry able to translocate into the and fry able to translocate into the intestinal enterocytes?intestinal enterocytes?

Scanning electron m icroscopy m icrograp h of the m idgut of A tlantic salm on (Salm o salar L .). M V – m icrovilli, E – enterocyte, L – lip id droplet, B M – basalm em brane, L P – lam ina propria. X = 1000. A fter R ingø (unp ublished data ).

Endocytosis of bacteria in enterocytes in posterior part of the Endocytosis of bacteria in enterocytes in posterior part of the hindgut hindgut of a 14of a 14--dayday--old herring (old herring (Clupea harengusClupea harengus L.) larva. Bacteria appear to L.) larva. Bacteria appear to be interiorised in a membranebe interiorised in a membrane--bound vacuoles wherein they undergo bound vacuoles wherein they undergo digestion. digestion. After Hansen and Olafsen (1999)After Hansen and Olafsen (1999)

Endocytosis of bacteria (arrows) in posterior to the rudiment Endocytosis of bacteria (arrows) in posterior to the rudiment of the stomach in 5of the stomach in 5--days post hatched turbot larvae. days post hatched turbot larvae.

Mitochondria (arrowheads). MVMitochondria (arrowheads). MV--microvilli. microvilli. After MacQueen After MacQueen Leifson et al. (2003)Leifson et al. (2003)

Pleomorphic intracellular bacteria in the gut of 22 days old larPleomorphic intracellular bacteria in the gut of 22 days old larvae of great vae of great scallop (scallop (Pecten maximusPecten maximus). The bacteria (arrows) are ). The bacteria (arrows) are intravacuolarintravacuolar, elongated, , elongated,

branched and without nuclear areas. branched and without nuclear areas. After Torkildsen et al. (2005)After Torkildsen et al. (2005)

Transmission electron microscopy (TEM) micrograph of the foregutTransmission electron microscopy (TEM) micrograph of the foregut of spotted of spotted wolffish fry. Bacterial profiles (arrows) are seen at various dewolffish fry. Bacterial profiles (arrows) are seen at various depths within pths within

enterocytes, and intraepithelial lymphocytes (IEL) are seen betwenterocytes, and intraepithelial lymphocytes (IEL) are seen between een enterocytes near the basal lamina. mventerocytes near the basal lamina. mv--microvilli; Vmicrovilli; V--vacuole; Nvacuole; N--nuclei; nuclei;

LPLP--lamina propria. X=7500. lamina propria. X=7500.

TEM micrograph showing bacteriaTEM micrograph showing bacteria--like profiles (arrows) in the subapical like profiles (arrows) in the subapical cytoplasm of midgut enterocytes of spotted wolffish fry. cytoplasm of midgut enterocytes of spotted wolffish fry.

MVMV--microvilli; mmicrovilli; m--mitochondria. X=15000. mitochondria. X=15000.

TEM micrograph of enterocytes in the hindgut of spotted wolffishTEM micrograph of enterocytes in the hindgut of spotted wolffish fry. fry. Several bacterial profiles (arrows) are seen. Several bacterial profiles (arrows) are seen.

mvmv--microvilli; Nmicrovilli; N--nuclei; LPnuclei; LP--lamina propria. X=3750. lamina propria. X=3750. After RingAfter Ringøø et al. (2003)et al. (2003)

a

CONCLUSIONS. CONCLUSIONS. IntracellularIntracellularttranslocation of indigenous bacteriaranslocation of indigenous bacteria

The indigenousindigenous gut bacteria are able to translocate in the gastrointestinal tract of fish larvae, larval scallop and fish fry.

Endocytosis into cytoplasm, and encapsulation by membrane vacuole are involved in intracellular translocation.

No cell damage has been observed when the indigenousindigenous bacteria translocate in the intestine.

IndigenousIndigenous gut bacteria has not been demonstrated in lamina propria.

PARACELLULAR TRANSLOCATIONPARACELLULAR TRANSLOCATION

This mechanism requires either loss ofenterocytes or loosening of their of celljunctions. Paracellular translocation has beenreported for Yersinia pseudotuberculosisYersinia pseudotuberculosis inepithelial cells and is promoted by β1-integrinmediated effects of tight junctions.

Has paracellular translocation been reportedHas paracellular translocation been reportedin the gastrointestinal tract of larvae and fry?in the gastrointestinal tract of larvae and fry?

TEM micrograph of enterocytes in the hindgut of spotted wolffishTEM micrograph of enterocytes in the hindgut of spotted wolffish fry exposed fry exposed to to V. anguillarumV. anguillarum. Notice the severe necrosis. Notice the severe necrosis--like damage to enterocytes, like damage to enterocytes,

increased intracellular gap (arrowheads) and damage in laminaincreased intracellular gap (arrowheads) and damage in laminapropria (LP). mvpropria (LP). mv--microvilli; AFmicrovilli; AF--apical microvillous brush border fragment; apical microvillous brush border fragment;

LL--lipid droplets; Nlipid droplets; N--nuclei; Vnuclei; V--vacuole; bacteriavacuole; bacteria--like profile (arrow). The bacterial like profile (arrow). The bacterial profile is paracellular rather than intracellular. X=3750. profile is paracellular rather than intracellular. X=3750.

Occurrence and translocation of pathogenic Occurrence and translocation of pathogenic bacteria in the GI tract of larvae and frybacteria in the GI tract of larvae and frySeveral studies have isolated Aeromonas salmonicidaAeromonas salmonicida the causative agent of furunculosis from the GI tract of larvae and fry by dilution plate technique.No study has been carried out demonstrating A. salmonicidaA. salmonicida in intestinal enterocytes of larvae and fry, but the bacteria has been reported in intestinal enterocytes of adult Arctic charr.

Vibrio anguillarumVibrio anguillarum the causative agent of vibriosis has been isolated from the intestine of Atlantic cod and turbot larvae.V. anguillarumV. anguillarum has been reported in the lamina propria (hindgut) of turbot larvae.

Endocytosis of Vibrio salmonicida and Vibrio fischeri have been reported in the foregut of Atlantic cod larvae.

SemiSemi--thin sections from Atlantic halibut larva challenge with thin sections from Atlantic halibut larva challenge with Aeromonas salmonicidaAeromonas salmonicida stained with Giemsa. Bacterialstained with Giemsa. Bacterial--like like structures can be seen (arrow). structures can be seen (arrow). After Bergh et al. (1997)After Bergh et al. (1997)

Localisation of Localisation of Vibrio anguillarum Vibrio anguillarum by GFP in zebrafish following by GFP in zebrafish following immersion infection. After 2 h of infection: A, entry of immersion infection. After 2 h of infection: A, entry of V. anguillarumV. anguillarum

through the mouth into the GI tract; B, localisation of through the mouth into the GI tract; B, localisation of V. anguillarumV. anguillarum in in the intestine. the intestine. After O`Toole et al. (2004)After O`Toole et al. (2004)

Vibrio anguillarumVibrio anguillarum infection in turbot larvae after oral infection in turbot larvae after oral challenge. Immunohistochemical staining showing what appears challenge. Immunohistochemical staining showing what appears to be release of to be release of V. anguillarumV. anguillarum (arrowhead) from an endocyte (arrowhead) from an endocyte

into lamina propria. Bar=10 into lamina propria. Bar=10 µµm. m. After Grisez et al. (1996)After Grisez et al. (1996)

INTESTINAL CELL DAMAGE CAUSED INTESTINAL CELL DAMAGE CAUSED BY PATHOGENIC BACTERIABY PATHOGENIC BACTERIA

Pathogenesis, the ability of microorgansims to initiate disease, includes entry, colonisation and growth of microorgansims in the host, resulting in changes in host functions that damage the host. A pathogen must usually gain access to host tissues and multiply before damage can be done.

What about fish pathogenic bacteria? Do they What about fish pathogenic bacteria? Do they cause cell damage in different parts of the cause cell damage in different parts of the digestive tract of larvae and fry?digestive tract of larvae and fry?

TEM micrograph of an enterocyte in the TEM micrograph of an enterocyte in the foregutforegut of spotted wolffish fry of spotted wolffish fry exposed to exposed to Vibrio anguillarumVibrio anguillarum. Notice the disintegrated microvilli (MV). Intact . Notice the disintegrated microvilli (MV). Intact

tight junction (TJ) was observed at the apical part of the entertight junction (TJ) was observed at the apical part of the enterocyte, but ocyte, but damage was observed in the lower part of the enterocyte. Bdamage was observed in the lower part of the enterocyte. B--bacterial like bacterial like

profile; Nprofile; N--nuclei; Vnuclei; V--vacuole. X=7500. vacuole. X=7500.

TEM micrograph of enterocytes in the TEM micrograph of enterocytes in the hindguthindgut of spotted wolffish fry exposed of spotted wolffish fry exposed to to Vibrio anguillarumVibrio anguillarum. Notice the disintegrated microvilli (MV) and a detached . Notice the disintegrated microvilli (MV) and a detached enterocyte (arrow). Cell damage is observed at apical part of thenterocyte (arrow). Cell damage is observed at apical part of the enterocyte.e enterocyte.

NN--nuclei; Vnuclei; V--vacuole. X=3750. vacuole. X=3750.

CONCLUSIONS: MECHANISMS INVOLVED IN CONCLUSIONS: MECHANISMS INVOLVED IN TRANSLOCATION OF BACTERIA IN THE TRANSLOCATION OF BACTERIA IN THE DIGESTIVE TRACT OF LARVAE AND FRYDIGESTIVE TRACT OF LARVAE AND FRY

INTRACELLULAREndocytosis into cytoplasm Hansen and Olafsen (1990)

Hansen and Olafsen (1999) MacQueen Leifson et al. (2003)Ringø et al. (2003)The present study

PARACELLULARThis mechanism involve loosening of cell junctions, and has only beenreported in one study of spotted wolffish fry

The present study

INTESTINAL CELL DAMAGELoss of cellular integrity. Spotted wolffish fry*midgut Ringø et al. (2003)*foregut and hindgut The present study

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Thanks to Ms. Turid KainoMs. Turid Kaino and Ms. PremasanyMs. PremasanyKanapathippillaiKanapathippillai for their excellent technicalassistance. The authors are grateful to Drs.Drs.Bergh, Grisez, Hansen, MacQueen Leifson,Bergh, Grisez, Hansen, MacQueen Leifson,O`Toole O`Toole and TorkildsenTorkildsen for providing theirphotographs. Financial support from ”AquacultureProtein Centre” and Institute of Marine Researchfor ER are gratefully acknowledged.