Veterinary Immunology and Immunopathology, 18 (1988) 181-193 181 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Alterations in the Peripheral Lymphoid Organs and Dif ferent ia l Leukocyte Counts in Saprolegnia- Infected Brown Trout, Salmo t ru t ta fario

F. ALVAREZ, B. RAZQUIN, A. VILLENA, P. LOPEZ FIERRO and A. ZAPATA ~

Departamento de Biologia Celular y Anatomia, Universidad de Ledn, 24071 Ledn (Spain) ~Departamento de Biolog[a Celular, Facultad de Biologia, Universidad CompIutense, 28040 Madrid (Spain)

(Accepted 1 July 1987)

ABSTRACT

Alvarez, F., Razquin, B., Villena, A., Ldpez Fierro, P. and Zapata, A., 1988. Alterations in the peripheral lymphoid organs and differential leukocyte counts in Saprolegnia-infected brown trout, SaImo trutta fario. Vet. Immunol. Immunopathol., 18: 181-193.

The number of circulating leukocytes and the structure of splenic and renal lymphoid tissue were comparatively analysed in healthy and Saprolegnia-infected wild brown trout, Salmo trutta fario. Sick trout showed lymphopenia, mainly due to decreased numbers of circulating small lym- phocytes, and heterophilia. The splenic and renal lymphoid tissue of infected trout exhibited similar changes, consisting of cellular depletion, lymphoid cell degeneration, and vascular altera- tions with blood vessel enlargement and hypertrophy of sinusoidal endothelial cells. Furthermore, the endothelial cells in the spleen and kidney of the infected trout contained cytoplasmic vesicles filled with material of possible fungal origin. The absence of a reticular sheath was also evident in the splenic ellipsoids. These results suggest some immunodepression in Saprolegnia-infected trout which might favour the course of the disease.

INTRODUCTION

Saprolegnia diclina is a frequent opportunistic or primary infectious agent in salmonids, which in recent years has produced high mortality rates in the wild brown trout populations of Spanish rivers. Previous studies of saproleg- niasis in salmonids were focused on: (1) frequency and distribution patterns of the disease on the fish body surface (Richards and Pickering, 1978); (2) histopathology of the skin lesions induced by the fungus ( Pickering and Rich- ards, 1980) ; (3) changes in the serum parameters of infected fish (Richards and Pickering, 1979) ; and (4) predisposing and protecting factors associated with the disease ( Pickering and Willoughby, 1982a, b). Although a weak lyre- phocyte infiltration in the skin lesions has been detected in Saprolegnia-in-

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fected fishes (Wolke, 1975; Pickering and Richards, 1980) and precipitins against Saprolegnia antigens were found in the serum of Salmo salar (Hodkin- son and Hunter, 1970), a systematic study on the possible role of the fish lymphoid system in host protection has not been made. In the present work, we have comparatively analysed the proportions of circulating leukocytes and the morphology of splenic and renal lymphoid tissue in healthy and Saproleg- nia-infected wild brown trout, Salmo trutta fario, in order to test the immune status of infected fishes and its possible influence on the course of the disease.

MATERIALS AND METHODS

Animals

Healthy and Saprolegnia diclina type 1-infected wild brown trout, Salmo trutta fario, were caught by electrical fishing in several rivers of Ledn (Spain) from January to April in 1984 and 1985. Fish were anaesthetized with MS 222 (Sandoz), weighed, and their ages determined by scalimetry. They were 3-7 years of age. Four-year-old male and female fish predominated among the healthy population studied, whereas sick males were principally 5 years old, and sick females 3-6 years old.

Differential leukocyte counts

Smears of peripheral blood were fixed in methanol and stained according to the Giemsa-May Grfinwald method. Three-hundred leukocytes, including small and large lymphocytes, heterophils, eosinophils, basophils and monocytes, were counted for each trout on three smears, and the mean and standard deviation calculated. A statistical analysis to determine significant differences was car- ried out using the Student's "t" test with a significance of P ~< 0.05.

Histological techniques

The spleen, pronephros and mesonephros as well as pieces of skin lesions were aseptically removed and routinely processed for light and electron mi- croscopy. Paraffin sections from Bouin's liquid-fixed tissues were stained with Alcian blue-Masson's haemalum-Picroindigo carmine. For electron micros- copy, small tissue pieces were fixed in 2.5% glutaraldehyde buffered to pH 7.3, postfixed in 1% osmic tetroxide and embedded in Araldite (Durcupan). Ultra- thin sections were double stained with lead citrate and uranyl acetate and ob- served in a JEOL 100C electron microscope. Semithin sections stained with an alkaline solution of toluidine blue were used to select the best areas for the ultrastructural study.

RESULTS

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Since no other infectious agents were isolated from the infected fish, Sap- rolegnia diclina type I was considered as the primary, direct causative pathogen of the disease. Haematological parameters and lymphoid tissues in ecto- thermic vertebrates undergo cyclic, seasonal variations (Zapata et al., 1983).

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Fig. 1. Differential leukocyte counts in healthy (fine stipples) and Saprolegnia-infected (large stipples) trout caught from January to April. Vertical bars represent the standard deviation of the means. (a) Proportion of total circulating lymphocytes. Significant differences between the two groups of fishes occur in January and April (P<0.05). (b) Proportion of small lymphocytes. Significant differences occur in January and February (P~< 0.05). (e) Proportion of large lym- phocytes. Significant differences occur in February (P ~ 0.05 ). (d) Proportion of circulating het- erophils. Sick trout exhibited significant heterophilia in all months of the study (P ~< 0.05 ). Note the different scales.

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Fig. 2. (a) Moderate development of the splenic lymphoid tissue of a healthy trout. (b) Spleen of a Saprolegnia-infected trout showing decreased lymphoid elements plus pycnotic cells. Ellipsoidal blood vessels are prominent in the sick fishes (arrows). Both fishes were caught in February.

Because of this, the present study considers only differences between healthy and infected trout collected in the same month.

Differential leukocyte counts

No statistical differences in leukocyte counts were found in either healthy or sick trout between males and females. Major alterations in the leukocyte counts of infected trout consisted of lymphopenia and heterophilia, while sig- nificant differences were not observed in eosinophil, basophil and monocyte numbers when both groups of t rout were considered. At monthly intervals, the total circulating lymphocyte population was lower in sick trout than in healthy fish (Fig. l a ) with significant differences occurring in January and April. This decrease was mainly due to significantly decreased circulating small lympho- cytes in sick trout, especially in January and February ( Fig. lb ), although they contained higher numbers of large lymphocytes than healthy fishes, with sig- nificant differences in February (Fig. l c ) . On the other hand, the infected trout exhibited significant heterophilia in all months of the study (Fig. ld ) .

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Fig. 3. (a) Splenic white pulp of a Saprolegnia-infected trout caught in January. Note the de- creased number of cells, the pycnotic elements (arrows) and the macrophages (M) containing engulfed cell debris. PC, pigment cell; A, arteriole; L, Lymphocyte. (b) Semithin section of the spleen of a Saprolegnia-infected trout caught in April. Note the enlarged sinusoids (S) and el- lipsoids (arrows).

Changes in the lymphoid tissues

As in the differential leukocyte counts, no apparent sexual dimorphism was observed in the morphology of splenic and renal lymphoid tissues, either in healthy or sick trout. The spleen of healthy brown trout consisted of poorly defined areas of red pulp, formed by cell cords arranged among blood sinusoids, and some little-developed lymphoid accumulations (Fig. 2a ). In addition, blood ellipsoids with typical reticular sheaths occurred in the splenic parenchyma (Fig. 5a). The spleen of Saprolegnia-infected trout showed less lymphoid tis- sue (Fig. 2b ) than that of healthy fish in all months of the study (Fig. 2a), as well as numerous pycnotic lymphocytes (Fig. 2b ) and large macrophages, which contained erythrocytes and cell debris ( Fig. 3a). The blood vessels in the spleen of infected trout also exhibited remarkable alterations consisting of enlarged sinusoids and ellipsoids (Fig. 3b), hypertrophy of the sinusoidal endothelial cells (Fig. 4) and diminution of the reticular cells in the ellipsoidal sheaths

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Fig. 4. Hypertrophic endothelial cells (EC) of the splenic sinusoids of a Saprolegnia-infected trout. The endothelial cells contain cytoplasmic vesicles (arrows) with an electron-dense content and a lucent peripheral halo. Dense bodies (arrow heads). Inset: High magnification of a cyto- plasmic vesicle showing its electron-dense content. × 55 000.

(Fig. 5b). Furthermore, the endothelial cells of the red pulp sinusoids con- rained cytoplasmic vesicles with a striking electron-dense content (Fig. 4).

The renal lymphoid tissue occurred principally in the pronephros, where the renal tubules and glomeruli are lacking, and also in the intertubular paren- chyma of the mesonephros. In any case, its histology was similar in both lo- cations, as were the modifications observed in the Saprolegnia-infected animals. The renal lymphoid tissue in healthy trout was formed by cell cords (where mature and immature blood cells of different lineages were housed), and en- larged blood vessels, most of which were sinusoidal in nature (Fig. 6a). In all months of the study morphological changes consisted of a decrease in lympho- haematopoietic tissue (Fig. 6b) with abundance of macrophages and modifi- cations in the blood sinusoids, whose endothelial cells underwent hypertrophy (Fig. 7). The hypertrophied endothelial cells contained cytoplasmic vesicles with an electron-dense content and an electro-lucent halo (Fig. 7), similar in morphology to those described in the splenic endothelial cells in sick trout (Fig. 4), although they were more abundant in the renal sinusoids than in the splenic ones.

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Fig. 5. (a) Splenic ellipsoids of a healthy trout. Cuboidal endothelial cells (E) are surrounded by a closely packed sheath of reticular cells ( RC ) which contain cytoplasmic dense bodies. (b) Splenic ellipsoid of a Saprolegnia-infected trout. Note the decreased numbers of reticular cells in the sheath surrounding the blood vessels. MC, mast cell; E, endothelium.

The morphology of these cytoplasmic vesicles ultrastructurally resembles the so-called "dense-body vesicles" present in the cytoplasm of fungal hyphae. Furthermore, the electron-dense content of these vesicles was morphologically similar to that found in the fuzzy wall coat of the Saprolegnia hyphae invading the trout skin.

DISCUSSION

The present study has shown, for the first time, that Saprolegnia-infected Salmo trutta fario exhibited altered circulating leukocyte counts and remark- able morphological changes in the splenic and renal lymphoid tissue. Lympho- penia in fish has been related to increased pituitary activity with a subsequent elevation of circulating corticosteroids (Weinreb, 1958; McLeay, 1973a, b; Pickering, 1986). Both chronic and acute stress, which in fishes involves in- creased circulating corticosteroids (Pickering, 1984) deplete the number of circulating lymphocytes (McLeay, 1975; Peters et al., 1980; Pickering et al., 1982; Tomasso et al., 1983; Pickering, 1986 ). Nevertheless, Pickering and Pot-

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Fig. 6. (a) Pronephric lympho-haematoipoietic tissue of a healthy brown trout caught in April, showing moderate numbers of lymphoid elements and some scattered melanomacrophages (arrows). Fig. 6. (b) Lympho-haematopoietic tissue in the pronephros of a Saprolegnia-infected trout caught in April, which contains only a few lymphoid cells. Melano-macrophages (arrows) and enlarged sinusoids (S) are evident.

tinger (1985) recently reported that chronic elevation of plasma cortisol has no effect on the number of circulating lymphocytes in Sahno trutta. Thus, the lymphopenia observed in Saprolegnia-infected Salmo trutta ratio might be ex- plained by the elevated levels of plasma cortisol associated with the disease. In this sense, preliminary data in our laboratory clearly demonstrate higher levels of cortisol in the infected than in control trout. In agreement with this, Pick- ering and Christie (1981) reported elevated cortisol levels in serum of Sapro- legnia-infected brown trout. On the other hand, heterophilia is a common phenomenon described in other fish diseases and under different experimental conditions (Finn and Nielsen, 1971; Hines and Spira, 1971). However, het- erophilia has also been related to processes including lymphopenia, such as stress or ACTH administration (Weinreb, 1958; Slicher, 1961 ).

The cellular depletion of the splenic and renal lymphoid tissues in Saproleg- nia-infected trout might also be mediated by the lymphocytotoxic activity of the elevated corticosteroid levels (Ellis, 1981 ) found in the plasma of infected

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Fig. 7. Hypertrophic endothelial cells (EC) in renal sinusoids of a Saprolegnia-infected trout. The endothelial cells contain cytoplasmic vesicles with a core of electron-dense material (arrows) and numerous dense bodies. Inset: High magnification of a cytoplasmic vesicle showing its elec- tron-dense content. × 55000.

trout. Decreased amounts of lymphoid tissue with cell lysis have been reported in the spleen and kidney of cortisone-treated teleosts (Rasquin, 1951; Chil- monczyk, 1982; Ghoneum et al., 1986). Likewise, Tamura and Honma (1974, 1975) found that the maximum lymphopoietic activity in the spleen and pro- nephros of Luciogobius guttatus and Chaenogobius urotaenia coincided with the minimal development of the interrenal tissue. In addition, Anderson et al. (1982) demonstra ted a decreased number of splenic plaque-forming cells, cir- culating antibody titers and splenic lymphocytes after corticosteroid injection in Salmo gairdneri. In any case, whether the suggested steroid-dependent lym- phocyte depletion found in Saprolegnia-infected trout is a cause or an effect of the disease is difficult to resolve conclusively.

Apart from the reported lymphocyte variations, the lymphoid organs of Sap- rolegnia-infected brown trout showed important modifications in their phago- cytic components and vascular system. A large increase in the number of macrophages, apparently related to the intense erythrocyte engulfment, was frequently observed in infected trout. Massive destruction of erythrocytes in the spleens of fishes supporting infective processes has generally been related

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to the release of erythrocyte membrane-alterating toxins (Riddle et al., 1981; Stevenson and Allan, 1981). Although Nolard-Tintigner (1973) was unable to demonstrate toxins in Saprolegnia infections, his results require new con- firmation. Moreover, a secondary bacterial infection in Saprolegnia-infected trout cannot be absolutely ruled out.

The vascular alterations in Saprolegnia-infected trout included enlarged vessels, lack of reticular cells in the splenic ellipsoidal walls, and hypertrophic sinusoidal-lining cells containing striking cytoplasmic vesicles. Enlarged ves- sels might reflect the osmoregulatory problems caused by skin destruction after fungus implantation, which included serum osmotic pressure with decreasing concentrations of K +, Ca 2+, Mg ~+, and total proteins (Richards and Picker- ing, 1979). On the other hand, we have no clear explanation for the lack of sheath reticular cells in splenic ellipsoids of infected trout. However, two hy- potheses can be postulated: (1) phagocytosis of fungal materials leads to cell death; and ( 2 ) saprolegniasis elicits a relocation of macrophages in the lymph- oid organs. Abundance of pycnotic cells to be phagocytosed could support the latter hypothesis. Moreover, we did not observe the presence of vesicles ultra- structurally similar to those described in the sinusoidal endothelial cells in the cytoplasm of circulating macrophages, nor in those housed in the splenic or renal parenchyma. In any case, as fish sheath reticular cells are involved in the trapping and processing of non-antigenic and antigenic materials (Secombes and Manning, 1980; Lamers and De Haas, 1985; Lamers and Parmentier, 1985 ), lack of this cell type might reflect the immunodepressed status of Saprolegnia- infected trout.

Although previous studies demonstrated neither the presence of Saprolegnia hyphae in the inner tissues of infected fish nor the presence of systemically transmitted toxins in saprolegniasis (Pickering and Willoughby, 1982b), phagocytosis of detached fungal electron-dense materials appears to occur in the hypertrophic endothelial cells of the splenic and renal sinusoids in sick trout. They contained cytoplasmic vesicles with an electron-dense content ul- trastructurally similar to that found in either the cytoplasmic dense-body ves- icles or in the fuzzy coat of the wall of Saprolegnia hyphae. Cytoplasmic dense- body vesicles were also reported in the hyphae of Phytophthora (Powell and Bracker, 1977; Aliaga and Ellzey, 1984 ) and Saprolegnia ferax ( Beakes, 1980). They contain lipids and fl-l,3 glucans which play a role in fungal wall forma- tion (Armbruster, 1982). On the other hand, the presence of these vesicles in the sinusoidal endothelial cells might also be the result of the phagocytosis of substances resulting from the fungal chymotrypsin-like enzymatic activity, or the enzyme itself, which mediated the penetration of the host tissues by Sap- rolegnia hyphae (Peduzzi et al., 1976; Peduzzi and Bizzozero, 1977). The hy- pertrophic aspect of the sinusoidal endothelial cells would appear to indicate that these cells are unable to metabolize the material contained in the cyto- plasmic vesicles, or that this material has a toxic effect on the endothelial cells.

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In any case, because the sinusoidal endothelial cells of the spleen and kidney form a part of the reticulo-endothelial system of teleosts, saprolegniasis might induce a RES-blockade or destruction resulting in immunodepression in the sick trout. Moreover, altered functions of the splenic and renal sinusoids might also explain osmotic problems leading to fish death.

In summary, our morphological results indicate that saprolegniasis might elicit immunodepression in wild brown trout. Further studies are, nevertheless, necessary to clarify the role played by this phenomenon in the course of dis- ease. In this respect, experiments are underway in our laboratory to analyse the possible relationships between seasonal changes in teleost lymphoid or- gans and seasonal major incidence of saprolegniasis in wild brown trout, the pathogenic effects of the materials released from the fungus, and the immu- nological status of the diseased fishes.

ACKNOWLEDGEMENTS

This study was supported, in part, by a grant from the "Consejeria de Agri- cultura de la Junta de Castilla y LeSn" to the research project "Acuicultura LeSn 84". Brown trout were generously provided by the "Instituto Provincial de Montes, Caza, Pesca y ProtecciSn de la Naturaleza de LeSn".

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