Bacterial Infection: Yersinia Ruckeri Infection in Salmon Fry

1. Bacterial infection - example 2:Yersinia ruckeri infection in salmon fry. Case 1: This spleen also initially looks to be disrupted and disorganized, but this is mostly due to a marked spleen reaction rather than necrosis, with few degenerate cells visible. This fish still has a good chance of survival. The two changes obvious at this magnitude are: more blood, and less cells in the non-lymphoid areas, visible as generally more spaces. S6 x 4

2. The kidney of this fish also shows increased interstitial spaces. This type of depletion has previously been discussed as a functional response of leukocyte and erythrocyte mobilization to meet the demands of infection, anoxia or other stress, resulting in at least a transient blood leukocytosis and / or increased PCV. A similar physiological depletion has occurred in the spleen. x 4

3. This can be seen on a more detailed look at another area of this spleen. – where are the ellipsoids? Just the vessels with a thin incomplete rim There are also a lot more leukocytes in the blood larger vessels (right) and diffusing through the ellipsoids (circled). S6 x 40 S6 x 4

4. Another near by thin walled vessel – apparently an ellipsoid with marked peripheral depletion, oedema, occasional pycnotic cell and one pigmented macrophage. S6 x 40

5. Ellipsoid reactions 3. Sub-acute reactions: • The increased debris or foreign material passing through the ellipsoid will increase phagocytic activity and lead to recruitment of more macrophages. • Infectious agents may also damage these cells. The toxins and interleukins released may increase blood flow and tissue spaces between the ellipsoid cells. • The increased blood flow and leukocyte mobilisation presumably aids macrophage recruitment to this site. • The active spleen becomes an important site for antigen trapping, and a perivascular lymphoid response may be seen.

6. Yersinia ruckeri Case 2: Another salmon fry with a later stage of the infection. Although the most obvious response is possibly the lymphoid response (the darker perivascular areas), the ellipsoids also show a response. The ellipsoids appear almost confluent because of depletion of the darker mature haemocyte cuffs and the reduction of interstitial congestion, though that is still present. S7 x 4o

7. Individual ellipsoids are slightly better visualised in this view (the congestion less so, though). S7 x 4o

8. The ellipsoids are more easily seen with increasing magnification, and variations in the thickness of the macrophage cuff more obvious. S7 x 10o S7 x20o

9. Some ellipsoid macrophage cuffs have increased from about 2 cells in thickness to about 4 or more. The macrophage nature is more obvious and the cells are generally more confluent, though many cells can still be seen flowing between these cells and leukocytes are common in the blood. S7 x40o

10. More ellipsoids of varying thickness. S7 x40o

11. Another ellipsoids with slightly increased macrophage cuff but dark mature cells. Note also the lymphocytes along the major vessel at left. S7 x40o

12. Yersinia ruckeri Case 3: A later stage of response in a slightly larger fish. Note the much larger ellipsoids, again consisting largely of macrophages without the dark peripheral cuff. There is also an obvious peri-arterial lymphoid response. Ex x 4

13. Ellipsoid pathology Antigen trapping and phagocytosis involves risk to the reticular cell and phagocyte. The following 2 examples show: • moderate damage to ellipsoids during a generally effective host response to Yersinia ruckeri • overwhelming infection with Vibrio anguillarum, a bacteria producing marked toxaemia.

14. Yersinia ruckeri Case 4: Early infection involving damage to some ellipsoid cells and subsequent precipitation of fibrin within the ellipsoid. At low magnification this appears similar to previous cases. However at higher magnification fibrin is visible within the ellipsoids Along with occasional polymorphs (white arrows) x4o

15. (Continued)Pycnotic cell fragments are also visible at high power ( ). This cell destruction appears to have elicited an inflammatory reaction within the ellipsoid with fibrin (pink arrow),active polymorphs (white arrows) and oedema. x 40o

16. (Continued)In some ellipsoids bacteria are visible, free and in phagocytic vacuoles. x100o Ex x 40

17. A more severe example of splenic ellipsoid damage, with infection of very young fry with a particularly toxic strain of Y. ruckeri. Note the widespread fibrinoid pink areas. Note the widespread bacteria within vessels, and in the adjacent tissues. E073642x20o E073642x4o E073642x10o x40o

18. Bacterial infection - example 3: Vibrio anguillarum inAtlantic salmon. The amount of blood is increased but the ellipsoids are clearly visible. How would you interpret these changes? Detailed ellipsoid structure is difficult to discern even at the higher magnification (x40 objective). No obvious peripheral cuff remains. There appear to be more ellipsoid cells (presumptive macrophages), but these are separated, distorted and sometimes pycnotic. Note too the interstitial distension and abundant pigment. The spleen shows considerable necrosis and loss of structure where “congestion” gradually becomes “haemorrhage” – a so-called “raspberry jam” spleen. This is spleen degeneration as well as reaction and is typical of terminal infection with highly toxic bacteria which destroy the filtration tissues where they are concentrated. S5 x4

19. Gross appearance of an Atlantic salmon with Vibrioanguillarum septicaemia. The spleen is swollen, dark and moist, reflecting the increasing vascular damage.

20. Another Atlantic salmon with V. anguillarum septicaemia. This spleen (and other tissues), have undergone more severe toxin induced vascular damage, with frank oedema and haemorrhage leaking into the abdominal cavity.

21. Lymphoid & other haematopoietic reactions - overview This section looks at the reactions of the lymphoid “white pulp”. We have already seen several spleens showing prominent peri-artial lymphoid tissue. Both the kidney and spleen are sites of lymphoid reactions and haematopoiesis in teleosts. • In many fish the spleen is regarded as a secondary site for these functions, though for some fish it is the only site, and for others, such as rainbow trout, it is the major site of erythropoiesis. (Ferguson HW. Systemic Pathology of Fish. A text atlas of comparative tissue responses in diseases of teleosts Iowa State University Press, Ames, 1989). • This is consistent with the finding that lymphocytes are first seen within the thymus, then the kidney, then the spleen (see PPT I). • The kidney is regarded as the main site of antibody production: the spleen is a secondary site for both T-cell and B-cell production.

22. Let us take a closer look at this salmon spleen showing an obvious lymphoid response, predominantly along the major vessels (T-cell dependent response). Clearly the major cells in the dark perivascular masses are normal active lymphocytes (small dark nuclei with very little cytoplasm). The nearby ellipsoids and fibrinoid interstitial areas confirm a general spleen reaction. S10 x 20o S10 x 10o S10 x 4o S2 x 10o

23. Another field from the same spleen. S10 x 20o

24. Another field from the same spleen, showing vessels surrounded by a lymphoid response in cross section: the response mimics a peri-ellipsoid response, except the reaction is generally around slightly larger vessels and the smaller ellipsoid complexes are not involved. S10 x 20o

25. Another Atlantic salmon, from a group with no definitive diagnosis, except for general signs of stress. This shows a similar perivascular lymphoid reaction, which is more obvious at higher magnification: Note that the ellipsoids (circled) are quite depleted of peripheral cuffs. E070613 x10o E070613 x4o

26. Contrast the preceding slides with this one, showing a much more generalised response round each of the ellipsoids, forming marked dark peripheral cuffs. This fish was from a group with reovirus infection, of unknown duration. It is probably that this fish has responded to a previous depletion of the peripheral cuffs during an earlier acute reaction. The ellipsoids are now close to normal, except for the increased size of the peripheral cuffs, which give an indication of a spleen response. In this case the cuff does appear to be largely composed of lymphoid cells, though it is not always possible to identify the lineage of immature cells in normal sections. Sreo x 10o Sreo x 4o

27. Certainly some fish in this group showed a lymphoid response - as demonstrated in this head kidney of another fish from this group.

28. Note that the dark zone of mostly mature cells also contains some large blast like cells, and cells suggesting mitosis, indicating active haematopoiesis. It is not always possible to identify individual cell lines, but in general the cell population involved appears mixed, both in type and level of maturity. This is another spleen from this cohort, showing more congestion, suggesting the fish is still in the active response phase. x 40o

29. Compare the previous slide with this one (also Atlantic salmon spleen). The ellipsoids are surrounded by a much more uniform population of dark cells…. Generally with the appearance of lymphoid cells – although several show a little basophilic cytoplasm suggestive of plasma cells (which would be expected of B-cell dependent reactions). x 20o x 100o x 20o x 10o

30. This spleen (which we will look at in more detail below), shows evidence of current haematopoietic activity with mitosis and putative immature erythrocytes (haemoglobin like staining of cytoplasm) around the ellipsoids. Overall the latter are active, but not large. S19 x 40o