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.     

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.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

A study of sequential histopathology of Trypanoplasma borreli (Protozoa: Kinetoplastida) in susceptible common carp Cyprinus carpio

The tissue response of common carp Cyprinus carpio to the kinetoplastid blood parasite Trypanoplasma borreli Laveran & Mesnil, 1901 was investigated during a laboratory infection of a highly susceptible carp line. With the development of the parasitaemia an increased proliferation of the lymphoid renal interstitial tissue was induced, which resulted in a progressive depression and deterioration of renal tubules. In heavily infected carp at Days 20 to 28 post inoculation (PI), a tubulonephrosis, a glomerulitis caused by a massive accumulation of leukocytes in glomerular capillaries, and large numbers of trypanoplasms in blood vessels and renal interstitium were observed. Corresponding with rising T. borreli numbers in the peripheral blood, splenic lymphocytes showed increasing proliferation rates, and the capillaries of the liver, gills, heart and intestine were infiltrated with lymphocytes and trypanoplasms. In heavily infected carp, congestion of liver sinusoids, focal necroses of hepatic tissue, extensive accumulations of erythrocytes in the spleen and in the blood marked anaemia were observed. These carp often showed abdominal distension, exophthalmus and swimming disorders described as 'sleeping sickness of carp'. Proliferation of cells from the interstitial lymphoid tissue of the kidney, which bears a close resemblance to the bone marrow of higher vertebrates, is considered a normal immune response of fish to antigen challenge. We here describe the unique case of a severe but ineffective immune reaction which results in the destruction of excretory renal structures. This has to be considered a severe disturbance of osmoregulation in affected carp, which, together with a decrease in oxygen uptake due to anaemia, is likely a major cause of death in these carp.     

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.     

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.     

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.     

Antibody levels in goats fed Toxoplasma gondii oocysts

Outbred goats were fed 10(5) Toxoplasma gondii oocysts and were monitored twice weekly for 8 wk for rectal temperature, clinical signs, parasitemia, and antibody levels by indirect fluorescence antibody test (IFAT), latex agglutination test (LAT), and indirect enzyme-linked immunosorbent assay (ELISA). After 8 wk, all goats were killed, and samples of heart, skeletal muscle, brain, lymph nodes, kidneys, and liver were bioassayed in mice. Anorexia, fever, and lethargy were observed from day 3 to day 7 postinfection (PI). Parasitemia was detected by bioassay in 50% of infected goats from day 7 to day 14 PI. Viable T. gondii organisms were isolated from all infected goats. Antibodies to T. gondii were detected in some animals on day 10 PI by IFAT and LAT and on day 14 PI by ELISA. The infected goats were seropositive on day 17 PI.     

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.     

Impaired Antigen Specific Responses and Enhanced Polyclonal Stimulation in Mice Infected with Ehrlichia muris

The immune status of BALB/c mice infected by intraperitoneal inoculation with Ehrlichia muris was examined. The level of E. muris infection in both peritoneal cavity and spleen was greatest at day 10 postinoculation (PI). Thereafter, the infection level was dramatically reduced while the organism persisted for up to 400 days PI. The greatest intraperitoneal infiltration of leukocytes, splenomegaly, and leukocytosis were observed on days 10, 15, and 20 PI, respectively. Infected mice developed marked hypergammaglobulinemia of IgG and IgM that peaked at day 20 PI; however, IgA plummeted at day 15 PI. Of IgG, G2a and G3 increased while G1 and G2b remained constant. Despite hypergammaglobulinemia, both IgG and IgM antibody titers against E. muris were very low throughout the 30-day study. Antibody development and plaque-forming cells against sheep red blood cells (SRBC) were abolished when the antigen was inoculated on day 10 PI. IgM antibody development against SRBC was more severely inhibited than IgG antibody development. However, when mice were immunized with SRBC prior to E. muris infection, antibody development against SRBC was not reduced. Delayed type hypersensitivity reaction to dinitrofluorobenzene was also maximally inhibited when the antigen was administered on day 10 PI. The IFN-γ level in the blood was maximal at day 10 PI. These results indicate that although the vigorous polyclonal activation and protective IFN-γ responses occurred by day 10 PI—which cleared most of the ehrlichial infection—antigen-specific immune stimulation was impaired primarily at the level of antigen-priming at peak parasitemia.     

Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.     

Trypanosoma evansi: concentration of 3-nitrotyrosine in the brain of infected rats

Nitric oxide (NO) is involved in many physiological processes, such as blood pressure control, neurotransmission, inhibition of platelet and neutrophil adherence, and the ability to kill tumor cells and parasites. The indirect determination of NO can be made by detection of 3-nitrotyrosine (3-NT) residues. The aim of this study was to measure the concentration of 3-NT in the brain of rats experimentally infected with Trypanosoma evansi. Twenty-four were inoculated intraperitoneally with cryopreserved blood containing 1 × 10⁶ trypomastigotes per animal. Twenty-four animals were used as negative controls and received 0.2 mL of saline by the same route. The experimental groups (group C and T) were established according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with six non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with six animals infected with T. evansi in each group. The animals were anesthetized with isoflurane and subsequently euthanized at the days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI). The brain was removed and dissected into cerebellum, cerebral cortex, striatum and hippocampus. Concentration of 3-NT in the brain was determined by Slot blot technique. At the day 3 PI no changes were observed in the concentration of 3-NT among the groups. There was a significant reduction (p < 0.05) of 3-NT concentration in the striatum and cerebellum at the days 5 and 10 PI, respectively. At the day 20 PI a significant increase (p < 0.05) of 3-NT was observed in the cerebellum, cerebral cortex and hippocampus from the infected animals. Therefore, T. evansi infection caused changes in the concentrations of 3-NT in the central nervous system (CNS), which may be related to clinical signs and infection management.     

Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection.

Mechanisms of parasite killing by eosinophils are widely studied and are often implicated in mediating resistance to parasitic infection, especially in conjunction with specific antibodies. Evidence for the eosinophil as an anti-parasite killer cell in vivo is limited and may not justify the belief that eosinophils engage and/or kill infective helminths. We reexamined this question in a mouse model of trichinosis in which antisera to eosinophils were previously used to show the requirement for eosinophils in resistance to this nematode. The current studies used mAb to IL-5 to suppress eosinophil levels in CF1 mice infected with Trichinella spiralis. In mice given a primary infection and injected with an isotype control mAb or left untreated, the medullary and peripheral blood eosinophil numbers peaked at 3 wk postinfection (PI) and returned to baseline levels by 4 wk PI. Peripheral blood eosinophil numbers in infected mice injected with anti-IL-5 were maintained at levels below those of uninfected normal mice through 4 wk of infection. Histologically, there was a prominent eosinophil accumulation in infected, untreated, or control-mAb-treated mice associated with nurse cell complexes containing infective juveniles in skeletal muscle at 3 and 4 wk PI. This was largely eliminated in mice treated with anti-IL-5 mAb. However, the number of muscle stage juvenile worms recovered 3 and 4 wk PI after acid pepsin digestion was unaffected by eosinophil depletion. Challenge infections, in which mice were infected at day 0 with 125 muscle stage worms and challenged at day 28 PI with 350 muscle stage worms, developed peak eosinophil numbers in bone marrow and peripheral blood 3 wk after primary infection and 2 wk after challenge infection in mice receiving either no treatment or control mAb. In challenged mice receiving anti-IL-5 mAb, medullary and peripheral blood eosinophil numbers remained at or below those of uninfected animals. Although all groups exhibited significant resistance measured as muscle stage worm burdens 56 days PI, eosinophil depletion did not affect resistance of muscle worm recovery. These results suggest that eosinophils are not essential in the control of T. spiralis in either primary or challenge infections of CF1 mice. This in vivo study illustrates the questionable value of in vitro killing assays to assign effector function to any single inflammatory cell type.     

Plasmodium gallinaceum: mechanisms of anemia in infected chickens

Erythrocyte labeling by random and cohort techniques was used to study erythrocyte survival in normal chickens and chickens infected with Plasmodium gallinaceum. Occurrence of erythrocyte destruction during the prepatent period was apparent in infected chickens by both techniques. Treatment with the antimalarials chloroquine and quinacrine not only cleared the circulation of parasites promptly but brought about an equally prompt cessation of disease-related erythrocyte destruction. Plasmodium gallinaceum infection caused a transitory decrease in blood volume at the time of rapid decrease in packed cell volume. The blood volume returned to preinfection values before the packed cell volume returned to normal. Parasitized erythrocytes were present in capillaries of the spleen, liver, and bone marrow during the entire prepatent period of the infection, thus providing a reasonable explanation for erythrocyte destruction observed in the absence of parasitemia during the prepatent period.     

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.     

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.     

Dynamics of cytokine production during coccidial infections in chickens: colony-stimulating factors and interferon

Abstract We assayed two classes of immunoregulatory cytokines, colony-stimulating factors (CSF) and interferon (IFN), during and immediately after a primary coccidial infection in chickens. Coccidial infection induces significant alterations in serum colony-stimulating activity (CSA) and these alterations immediately precede the characteristic biphasic leukocytosis. CSA rose sharply during the first 24 h post-inoculation (PI), but returned to control levels by 48 h PL. At this time, we detected an increase in peripheral blood leukocytes which peaked at 96 h PI. A second phase of CSA increase began 96 h PI and peaked at 120–144 h PI which again preceded the second phase of leukocytosis. We also examined the production of IFN during the first 20 days PI. Splenic T cells from Eimeria maxima -infected chickens produced significantly less IFN on day 5 PI compared to T cells from the coccidia-free controls. By days 10 and 15 PI, there was no significant difference in IFN production between the T cells of infected and non-infected chickens. However, by day 20 PI, IFN production by the T cells of the infected birds produced significantly more IFN than the control T cells. The results of our studies indicated the differential production of two different cytokines by chickens during and following a primary coccidial infection. Based on these experiments, CSF may be some of the first cytokines produced during an E. maxima -infection, while IFN may be one of the later cytokines produced.