Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.     

Cytopathological observations on renal tubule epithelium cells in common carp Cyprinus carpio under Trypanoplasma borreli (Protozoa: Kinetoplastida) infection

Cytological alterations in renal tubule epithelium cells of carp Cyprinus carpio infected with the blood flagellate Trypanoplasma borreli Laveran & Mesnil, 1901 were investigated during the course of a laboratory infection of a highly susceptible carp line. With the development of the parasitaemia, a hyperplasia of the interstitial renal tissue was induced, which resulted in a tubulus necrosis. Cytological changes were already seen in tubulus epithelium cells on Day 7 post injection (PI) of the parasite. The basilar invaginations of the cells fragmented and a swelling of mitochondria was noted. With increasing parasitaemia, on Days 14 and 21 PI, these changes progressed up to the loss of the basilar invagination and high amplitude swellings of mitochondria and deterioration of their internal membrane structures. Cells of the distal tubule segment reacted earlier and more rapidly than cells of the proximal tubule. The cytological alterations suggested a loss of function of the epithelum cells, which most likely resulted in impaired ionic and osmotic regulation of T. borreli-infected fishes. Our findings indicate that in response to the proliferation of the interstitial renal tissue cell structures of the renal tubule cells are altered quickly and in a progressive manner.     

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.     

Development of Trypanoplasma borreli (Mastigophora: Kinetoplastida) in the leech vector Piscicola geometra and its infectivity for the common carp, Cyprinus carpio

The development of Trypanoplasma borreli in the crop of the leech vector Piscicola geometra was characterized by significant changes in morphology. Immediately after ingestion by the leech, stumpy-shaped T. borreli predominated and numerous dividing specimens were found. This led to long and slender trypanoplasms near the end of the infection. The infection was terminated with complete digestion of the blood stored in the crop of the leech. The longest period of infection observed was 11 days. Trypanoplasma borreli was found only in the crop of the leech. At any time during the infection, T. borreli isolated from P. geometra cause a parasitemia when inoculated into parasite-free carp. There was no difference in morphology or infectivity among T. borreli isolated from various crop regions of P. geometra.     

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide,which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were tested in vitro. Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli-stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600 micromol l(-1) NO(-)(2)) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.     

The parasitemia of cloned Trypanoplasma borreli Laveran and Mesnil, 1901, in laboratory-infected common carp (Cyprinus carpio L.)

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 10(3) T. borreli/microliters blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/microliters blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 10(4) flagellates/microliters blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.     

Minor effect of depletion of resident macrophages from peritoneal cavity on resistance of common carp Cyprinus carpio to blood flagellates

Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.     

Modulation of carp (Cyprinus carpio) neutrophil functions during an infection with the haemoparasite Trypanoplasma borreli

Trypanoplasma borreli is an extracellular blood parasite of common carp (Cyprinus carpio) transmitted by fish-biting leeches. The infestation with this parasite in juvenile carp may range between 75% and 100%, especially in fish recovering from the first hibernation period. T. borreli is perfectly adapted to its prolonged survival in a cyprinid host. Elevated numbers of activated neutrophils in peripheral blood and tissues are reported during T. borreli infection, but in context of the disease, the direct reason for elevated neutrophil numbers and their role during the infection remain unclear. In this study, a quantitative transmigration system, permitting the harvest of highly pure (> or = 97%) neutrophil populations was applied to investigate the modulation of carp neutrophil functions during T. borreli infection. We demonstrate time-dependent kinetics of a serum-induced down-regulation of neutrophil chemotaxis and an up-regulation of ROS production during the course of infection. With highly pure neutrophil populations, we could show that this divergent alteration of neutrophil functions was neither caused by T. borreli metabolites nor by the parasite itself. Moreover, when added to highly purified neutrophils, parasite metabolites did not alter the leukotriene B4-induced neutrophil chemotaxis nor the Staphylococcus aureus-induced ROS production. We conclude that the haemoparasite T. borreli does not interact with neutrophils directly, but indirectly modulates their functions via serum factors induced by parasite interaction with other components of the immune system.     

Head kidney neutrophils of carp (Cyprinus carpio L.) are functionally modulated by the haemoflagellateTrypanoplasma borreli

In the present work responses of carp (Cyprinus carpio) head kidney-derived neutrophils to the blood parasite T. borreli, and the consequences of these responses for parasite survival and other host response mechanisms, were studied. In co-cultures of head kidney leucocytes (HKL) with viable and lysed T. borreli a prominent shape change of neutrophilic granulocytes towards increased size and complexity was observed. In addition, the longevity of neutrophils in vitro was prolonged in the presence of T. borreli antigens. In these cultures, neutrophils also exhibited an increased phagocytosis activity. An up regulation of the production of nitric oxide (NO) and reactive oxygen species (ROS) was observed in T. borreli- and mitogen-stimulated HKL cultures. However, addition of live, fluorescence-labelledT. borreli to previously stimulated HKL cultures, revealed neither killing nor phagocytosis of the parasite by activated neutrophils. Moreover, viable T. borreli, when added to HKL cultures of infected carp, reduced their phagocytosis activity and NO production. Supernatants of co-cultures between T. borreli and HKL also contained mediators, which suppressed a mitogen-induced proliferative response of peripheral blood leucocytes (PBL) in vitro. Thus, while T. borreli itself appeared not to be sensitive to responses of activated neutrophils, the flagellates interferes with the production of immunomodulatory signals of these cells, probably resulting in a partial immunosuppression, which may favour the parasite development in vivo.     

Morphologic and histochemical study of blood capillaries in boar testes: effects of abdominal cryptorchidism

BACKGROUND: Few data exist about the features of testicular microvasculature under normal and pathologic conditions. METHODS: The morphology and lectin affinity of testicular capillaries were examined in healthy boars and in unilateral and bilateral abdominal cryptorchid boars. RESULTS: The capillaries of scrotal testes contained a) the endothelial layer formed by two cells, b) the basal lamina constituted by collagen fibers and glycoconjugates with fucosyl, galactosyl, glucosyl, and neuraminic acid residues, and c) the pericyte layer formed by a single cell. These components participated in substrate exchange between blood and testicular tissue. The abdominal testes showed increased numbers of capillaries, which could exhibit a mature appearance, but also angiogenic or degenerative patterns. Angiogenesis was manifested in interstitial capillaries and was characterized by a) proliferation of endothelial cells, b) decreased thickness and decreased content of collagen fibers and glycoconjugates in the basal lamina, and c) lack of pericytes. Degenerative capillaries lay in association with seminiferous tubules and showed a) pyknotic endothelial cells; b) thickening, collagenization, and altered glycoconjugate content in the basal lamina; and c) increased development of pericytes. The angiogenesis of interstitial capillaries resulted in high vascular permeability, and the degeneration of intertubular capillaries led to defective substrate exchange between blood and seminiferous tubules. CONCLUSIONS: Unilateral abdominal cryptorchidism did not alter the morphology and function of capillaries in the scrotal testis. Unilateral and bilateral abdominal cryptorchidism resulted in increased numbers and abnormal morphology and function of capillaries in abdominal testes. The proliferation of interstitial capillaries correlated with the immaturity of Leydig cells, and the degeneration of intertubular capillaries correlated with the thickening of the lamina propria.     

Immunopathologic effects associated with Sarcocystis neurona-infected interferon-gamma knockout mice

Interferon-gamma knockout (IFN-gamma KO) mice were infected with Sarcocystis neurona merozoites to characterize the immunopathology associated with infection. By day 14 postinfection (PI), mice developed splenomegaly and lymphadenopathy, characterized by marked lymphoid hyperplasia with increased numbers of germinal centers. Additional histopathologic changes included increased extramedullary hematopoiesis, multifocal mixed inflammatory infiltrates in the liver, perivascular infiltrate of the liver and lung, and interstitial pneumonia. The total number of B-cell splenocytes (P < 0.05) and the percentage of B-cells increased on day 14 PI in the spleen and on day 28 PI in the lymph nodes (P < 0.05). By day 28 PI, the number of B-cell splenocytes decreased significantly. A non-subset-specific decrease in percentages of CD4 lymphocytes throughout all lymphoid organs was observed on day 14 PI. However, total CD4 and CD44/CD4 splenocytes increased significantly by day 28 PI. Early-activation CD8 lymphocytes were reduced in the blood and spleen, whereas memory CD8 lymphocyte percentages and total numbers were significantly increased. On the basis of the results, we propose that S. neurona-infected IFN-gamma KO mice are immunocompromised and unable to clear the infection. Thus, they develop B-cell exhaustion and a delayed, but sustained, increased number of memory CD4 and CD8 lymphocytes due to chronic antigen stimulation.     

Multiple acute temperature stress affects leucocyte populations and antibody responses in common carp, Cyprinus carpio L

Stress is a potential factor causing increased susceptibility of fish to pathogens. In this study, stress-induced immunological changes that may contribute to a decreased immune status were investigated. A 3 h drop in ambient water temperature of 9 degrees C was used as a relative mild and acute stress model for carp. Effects of this stressor on the dynamics of leucocyte populations were determined with specific monoclonal antibodies. The relative number of circulating B-lymphocytes in the total leucocyte population decreased significantly within 4 h after the onset of single or multiple cold shocks. This decrease was reversible, as B-lymphocyte numbers were restored within 24 h. Most probably, a redistribution of B-lymphocytes contributed to this phenomenon. In head kidney, an increase was measured in the relative number of B-lymphocytes. Granulocyte numbers showed opposite reactions: the percentage of granulocytes in the total leucocyte population nearly doubled in circulation and decreased significantly in the head kidney. This demonstrates that in vivo, a mild stressor differentially alters the distribution of leucocytes. In stressed carp, the percentage of apoptotic lymphocytes in blood is significantly higher compared with the unstressed animals. B-lymphocytes as well as Ig- lymphoid cells contributed to this increased apoptosis. Labelling of blood lymphocytes with a polyclonal antiserum against the glucocorticoid receptor also showed, besides B-lymphocytes, part of the Ig- lymphoid cell population to be glucocorticoid receptor positive. As the distribution of B-lymphocytes was substantially affected, the effect of temperature stress on T-lymphocyte-independent (trinitrophenyl-lipopolysaccharide) and T-lymphocyte-dependent (dinitrophenyl-keyhole limpet hemocyanin) humoral antibody responses was determined. Kinetics of the primary antibody response to the T-lymphocyte-independent antigen showed lower antibody titres in stressed carp during the onset of the immune response, implying a slower development of the antibody response against the T-lymphocyte-independent antigen.     

Effects of a parasite-induced nephritis on osmoregulation in the common carp Cyprinus carpio

Carp Cyprinus carpio infected with the haemoflagellate Trypanoplasma borreli undergo progressive nephritis associated with a destruction of approx. 40% of the nephric tubules. In an attempt to analyse the effect of the nephritis on the osmoregulation of affected carp, the clinical chemical properties of plasma and urine samples were analysed. Parasitised carp excreted greater amounts of electrolytes in their urine than uninfected carp which excreted highly diluted urine with an osmolality of about 10% of plasma osmolality. During the course of the infection, urine osmolality increased up to 26% of plasma osmolality by Day 21 post-infection (p.i.). The plasma:urine ratio of Na+ also increased, while concomitant losses of Mg2+, Ca2+, K+ and inorganic phosphate were less pronounced. Infected carp were able to maintain a normal solute balance in their plasma. Plasma hydration (indicated by decreased protein contents) occurred on Day 21 p.i. Our data indicate that in T. borreli-infected carp, reabsorption processes of the distal renal tubule were disturbed, while secretory and absorption processes in the proximal tubule appeared to be less affected. In addition, infected carp were able to compensate their increased ion losses, probably by (energy-consuming) active absorption processes. The energy budget of infected carp was additionally affected by a substantial direct consumption of plasma glucose by the parasite.     

Ontogeny and postnatal persistence of a strong suppressor activity in man

We report here on the ontogeny and postnatal persistence of an inhibited human immune response in which lymphocytes from human newborns strongly suppress the proliferation of adults' lymphocytes stimulated by phytohemagglutinin (PHA) or alloantigens in vitro. For this research we used a 2-way mixed lymphocyte culture (MLC) supplemented with PHA, with sex chromosomes acting as markers for dividing male and female cells, or alternatively a double chamber system. The proliferation of maternal lymphocytes was significantly suppressed by fetal lymphoid cells from the liver as early as the 8th week of gestation and by those from fetal blood at the 14th week or later during gestation. This strong suppressor activity persisted in 11-mo-old infants but usually disappeared after that time.     

Spontaneous interferon production by pulmonary leukocytes is associated with lentivirus-induced lymphoid interstitial pneumonia

Ovine lentiviruses share genome sequence, structural features, and replicative mechanisms with HIV, the etiologic agent of AIDS. A lamb model of lentivirus-induced lymphoid interstitial pneumonia, comparable to lymphoid interstitial pneumonia associated with pediatric AIDS, was used to investigate production of leukocyte-soluble mediators. Lentivirus-infected lambs and adult sheep with severe lymphoid interstitial pneumonia had significantly elevated levels of spontaneous interferon (IFN) production from pulmonary leukocytes compared with ovine lentiviruses-infected animals with mild or no lesions of lymphoid interstitial pneumonia or non-infected controls. However, peripheral blood mononuclear cells from lentivirus-infected lambs did not spontaneously release significant amounts of IFN. IFN production by pulmonary lymph node lymphocytes was enhanced in the presence of lentivirus-infected alveolar macrophages. Animals with lentivirus-induced disease and spontaneous IFN production had enhanced virus replication within tissues. The ovine lentiviruses-induced IFN had a m.w. of between 25,000 and 35,000 and was resistant to freeze/thawing procedures. The IFN activity was sensitive to trypsin and stable to low pH and heat. IFN with similar physical and biochemical properties was produced when ovine lentiviruses was added to control leukocyte cultures. IL-2 and PGE2 production and responses to mitogen by pulmonary lymph node lymphocytes of lentivirus-diseased lambs were not statistically different from control animals. Increased local production of IFN in lentivirus-infected host tissues may serve to accelerate the entry of leukocytes into virus-induced lesions promoting cell-mediated tissue damage and also provide increased numbers of cells for virus replication.     

Physiological compartmentation in gonadal tissue of the common carp (Cyprinus carpio L.)

Summary Physiological compartmentation in carp (Cyprinus carpio L.) gonads was investigated after intracardial injection of horseradish peroxidase (HRP) and two mouse anti-carp-sperm monoclonal antibodies.Immunohistochemistry revealed that a physiological barrier exists in carp testis for HRP and mouse IgG monoclonal antibody around the central lumina of the tubules in which the spermatozoa are located, but not around the cysts containing the precursor germ cells. The results with HRP were confirmed by electron microscopy. Mouse IgM monoclonal antibody did not penetrate the spermatogenic cysts. Probably because of its large size, it was almost exclusively located inside blood capillaries and only sparsely in the interstitial tissue.In the ovary, HRP was regularly distributed in the gonadal tissue, whereas the IgG antibody was predominantly localised on oogonia and early prophase oocytes. The results indicate that in contrast with the testis, no barrier around germ cells exists in the carp ovary.     

Lesions in mink (Mustela vison) infected with giant kidney worm (Dioctophyma renale).

Adult Dioctophyma renale occupied the enlarged renal pelvis of the right kidney of naturally infected mink. Lesions in the kidney parenchyma consisted of connective tissue proliferation in the interstitial tissue, tubular atrophy and fibrosis, and periglomerular fibrosis. The luminal surface of the renal pelvis wall was formed of numerous papillae covered with transitional epithelium. The nematodes in the lumen were bathed in an albuminous fluid containing red blood cells, epithelial cells and D. renale eggs. The left (uninfected) kidney was 60% larger than the left kidney of normal mink.