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

Transcription

1 Bacterial translocation and pathogenesis in the digestive tract of larvae and fry: What are the indigenous bacteria and pathogens doing? E. Ringø 1,2, R. Myklebust 3, T.M. Mayhew 4 and R.E. Olsen 2 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.

2 Schematic drawing of the digestive tract of spotted wolffish (Anarhichas minor Olafsen) fry; [1] oesophagus, [2] stomach, [3] foregut, [4] midgut, and [5] hindgut.

3 The three major routes of infection in fish are through; -skin - gills - gastrointestinal [GI]tract When discussing whether the gastrointestinal tract is a port of entry for pathogenic bacteria in fish. A important question arise: Are there any information about bacterial translocation and pathogenesis in the GI tract of larvae and fry?

4 TRANSLOCATION OF BACTERIA 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 translocation.

5 INDIGENOUS BACTERIA The intestinal microbiota in fish as in mammals are classified as autochthonous (indigenous) or allochthonous 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 and fry able to translocate into the intestinal enterocytes?

6 Scanning electron microscopy micrograph of the midgut of Atlantic salmon (Salmo salar L.). MV microvilli, E enterocyte, L lipid droplet, BM basalmembrane, LP lamina propria. X = After Ringø (unpublished data).

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

8 Endocytosis of bacteria (arrows) in posterior to the rudiment of the stomach in 5-days 5 post hatched turbot larvae. Mitochondria (arrowheads). MV-microvilli. After MacQueen Leifson et al. (2003)

9 Pleomorphic intracellular bacteria in the gut of 22 days old larvae of great scallop ( (Pecten maximus). The bacteria (arrows) are intravacuolar,, elongated, branched and without nuclear areas. After Torkildsen et al. (2005)

10 Transmission electron microscopy (TEM) micrograph of the foregut of spotted wolffish fry. Bacterial profiles (arrows) are seen at various depths within enterocytes, and intraepithelial lymphocytes (IEL) are seen between een enterocytes near the basal lamina. mv-microvilli; microvilli; V-vacuole; V N-nuclei; N LP-lamina propria. X=7500.

11 TEM micrograph showing bacteria-like profiles (arrows) in the subapical cytoplasm of midgut enterocytes of spotted wolffish fry. MV-microvilli; m-mitochondria. m mitochondria. X=15000.

12 TEM micrograph of enterocytes in the hindgut of spotted wolffish fry. Several bacterial profiles (arrows) are seen. mv-microvilli; microvilli; N-nuclei; N LP-lamina propria. X=3750. After Ringø et al. (2003) a

13 CONCLUSIONS. Intracellular translocation of indigenous bacteria The indigenous 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 indigenous bacteria translocate in the intestine. Indigenous gut bacteria has not been demonstrated in lamina propria.

14 PARACELLULAR TRANSLOCATION This mechanism requires either loss of enterocytes or loosening of their of cell junctions. Paracellular translocation has been reported for Yersinia pseudotuberculosis in epithelial cells and is promoted by β1-integrin mediated effects of tight junctions. Has paracellular translocation been reported in the gastrointestinal tract of larvae and fry?

15 TEM micrograph of enterocytes in the hindgut of spotted wolffish fry exposed to V. anguillarum.. Notice the severe necrosis-like damage to enterocytes, increased intracellular gap (arrowheads) and damage in lamina propria (LP). mv-microvilli; microvilli; AF-apical microvillous brush border fragment; L-lipid droplets; N-nuclei; N V-vacuole; V bacteria-like profile (arrow). The bacterial profile is paracellular rather than intracellular. X=3750.

16 Occurrence and translocation of pathogenic bacteria in the GI tract of larvae and fry Several studies have isolated Aeromonas 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. salmonicida in intestinal enterocytes of larvae and fry, but the bacteria has been reported in intestinal enterocytes of adult Arctic charr. Vibrio anguillarum the causative agent of vibriosis has been isolated from the intestine of Atlantic cod and turbot larvae. V. 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.

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

18 Localisation of Vibrio anguillarum by GFP in zebrafish following immersion infection. After 2 h of infection: A, entry of V. anguillarum through the mouth into the GI tract; B, localisation of V. anguillarum in the intestine. After O`Toole et al. (2004)

19 Vibrio anguillarum infection in turbot larvae after oral challenge. Immunohistochemical staining showing what appears to be release of V. anguillarum (arrowhead) from an endocyte into lamina propria. Bar=10 µm. After Grisez et al. (1996)

20 INTESTINAL CELL DAMAGE CAUSED BY 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 cause cell damage in different parts of the digestive tract of larvae and fry?

21 TEM micrograph of an enterocyte in the foregut of spotted wolffish fry exposed to Vibrio anguillarum.. Notice the disintegrated microvilli (MV). Intact tight junction (TJ) was observed at the apical part of the enterocyte, but damage was observed in the lower part of the enterocyte. B-bacterial B like profile; N-nuclei; N V-vacuole. V X=7500.

22 TEM micrograph of enterocytes in the hindgut of spotted wolffish fry exposed to Vibrio anguillarum.. Notice the disintegrated microvilli (MV) and a detached enterocyte (arrow). Cell damage is observed at apical part of the e enterocyte. N-nuclei; V-vacuole. V X=3750.

23 CONCLUSIONS: MECHANISMS INVOLVED IN TRANSLOCATION OF BACTERIA IN THE DIGESTIVE TRACT OF LARVAE AND FRY INTRACELLULAR Endocytosis into cytoplasm Hansen and Olafsen (1990) Hansen and Olafsen (1999) MacQueen Leifson et al. (2003) Ringø et al. (2003) The present study PARACELLULAR This mechanism involve loosening of cell junctions, and has only been reported in one study of spotted wolffish fry The present study INTESTINAL CELL DAMAGE Loss of cellular integrity. Spotted wolffish fry *midgut Ringø et al. (2003) *foregut and hindgut The present study

24 ACKNOWLEDGEMENTS Thanks to Ms. Turid Kaino and Ms. Premasany Kanapathippillai for their excellent technical assistance. The authors are grateful to Drs. Bergh, Grisez, Hansen, MacQueen Leifson, O`Toole and Torkildsen for providing their photographs. Financial support from Aquaculture Protein Centre and Institute of Marine Research for ER are gratefully acknowledged.