RESUMEN
With the present study, a culture system for successive life-cycle stages of the tapeworm Schistocephalus solidus was developed and this report documents for the first time, cultivation of the procercoid stage of S. solidus from eggs. Additionally we have transformed procercoids dissected from experimentally infected copepods and cultured procercoids into the early plerocercoid stage in vitro. Observations in the culture suggest that the coracidia can interact with their external environment and need no host specific stimuli, except for the components in the culture medium, for activation and hatching from the embryophore. Increasing the culture medium pH from 7.3 to 8.0 improved escape rates and frequencies of hook contractions, suggesting that the oncosphere may recognize and respond to environmental conditions along the host intestine. Procercoids in the culture did not stop growing indicating that conditions within the copepod may be important to limit growth and to induce transformation to plerocercoids. When procercoids are dissected from copepods and transferred to the culture, the outer tegument layers and cercomer starts to loosen. Comparison of the lectin staining of the loosened outer tegument layers and cercomer in procercoids dissected from copepods confirms that transitions of both, the oncosphere to procercoid and procercoid to plerocercoids, has taken place in the in vitro cultures.
Asunto(s)
Cestodos/crecimiento & desarrollo , Estadios del Ciclo de Vida , Animales , Copépodos , Lectinas/metabolismo , SmegmamorphaRESUMEN
In diverse animal species, from insects to mammals, females display a more efficient immune defence than males. Bateman's principle posits that males maximize their fitness by increasing mating frequency whereas females gain fitness benefits by maximizing their lifespan. As a longer lifespan requires a more efficient immune system, these implications of Bateman's principle may explain widespread immune dimorphism among animals. Because in most extant animals, the provisioning of eggs and a higher parental investment are attributes of the female sex, sex-role reversed species provide a unique opportunity to assess whether or not immune dimorphism depends on life history and not on sex per se. In the broad-nosed pipefish Syngnathus typhle, males brood and nourish the eggs in a ventral pouch and thus invest more into reproduction than females. We found males to have a more active immune response both in field data from four populations and also in an experiment under controlled laboratory conditions. This applied to different measures of immunocompetence using innate as well as adaptive immune system traits. We further determined the specificity of immune response initiation after a fully factorial primary and secondary exposure to a common marine pathogen Vibrio spp. Males not only had a more active but also a more specific immune defence than females. Our results thus indeed suggest that the sex that invests more into the offspring has the stronger immune defence.
Asunto(s)
Conducta Animal/fisiología , Caracteres Sexuales , Smegmamorpha/inmunología , Animales , Demografía , Femenino , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Interacciones Huésped-Parásitos , Recuento de Linfocitos , Linfocitos/fisiología , Masculino , Monocitos/fisiología , Reproducción , Estallido Respiratorio , Smegmamorpha/fisiología , Vibrio/inmunología , Vibriosis/inmunología , Vibriosis/veterinariaRESUMEN
Plerocercoids of the pseudophyllidean cestode Schistocephalus solidus infect the three-spined stickleback Gasterosteus aculeatus, with important consequences for the biology of host fish. Techniques for culturing the parasite in vitro and generating infective stages that can be used to infect sticklebacks experimentally have been developed, and the system is increasingly used as a laboratory model for investigating aspects of host-parasite interactions. Recent experimental laboratory studies have focused on the immune responses of hosts to infection, the consequences of infection for the growth and reproductive development of host fish and the effects of infection on host behaviour. Here we introduce the host and the parasite, review the major findings of these recent experimental infection studies and identify further aspects of host parasite interactions that might be investigated using the system.
Asunto(s)
Cestodos/fisiología , Infecciones por Cestodos/veterinaria , Enfermedades de los Peces/parasitología , Smegmamorpha/parasitología , Animales , Infecciones por Cestodos/parasitología , Interacciones Huésped-Parásitos , Estadios del Ciclo de VidaRESUMEN
To investigate and disentangle the role of major histocompatibility complex (MHC)-based 'good genes' and 'compatible genes' in mate choice, three-spined sticklebacks Gasterosteus aculeatus with specific MHC IIB genotypes were allowed to reproduce in an outdoor enclosure system. Here, fish were protected from predators but encountered their natural parasites. Mate choice for an intermediate genetic distance between parental MHC genotypes was observed, which would result in intermediate diversity in the offspring, but no mate choice based on good genes was found under the current semi-natural conditions. Investigation of immunological variables revealed that the less-specific innate immune system was more active in individuals with a genetically more divergent MHC allele repertoire. This suggests the need to compensate for an MHC-diminished T-cell repertoire and potentially explains the observed mate choice for intermediate MHC genetic distance. The present findings support a general pattern of mate choice for intermediate MHC diversity (i.e. compatible genes). In addition, the potentially dynamic role of MHC good genes in mate choice under different parasite pressures is discussed in the light of present and previous results.
Asunto(s)
Complejo Mayor de Histocompatibilidad/genética , Preferencia en el Apareamiento Animal/fisiología , Smegmamorpha/fisiología , Animales , Constitución Corporal , Femenino , Enfermedades de los Peces/fisiopatología , Genotipo , Granulocitos/citología , Recuento de Linfocitos , Masculino , Enfermedades Parasitarias en Animales/fisiopatología , Smegmamorpha/genética , Smegmamorpha/inmunología , Smegmamorpha/parasitologíaRESUMEN
The three-spined stickleback (Gasterosteus aculeatus) is an important model organism for investigations on the maintenance of polymorphism of the major histocompatibility complex (MHC) of vertebrates. Analysis of functional aspects of MHC diversity in stickleback would benefit from the availability of MHC specific reagents. Here we characterize antisera raised against recombinant fusion proteins of stickleback MHC class I alpha and class II alpha and beta. Western blot analysis using recombinant proteins confirmed the specificity of the antisera. In brain and muscle preparations, neither of the MHC types was detectable. High levels of each MHC receptor type were observed in gills and spleen and lower levels in head kidneys. In histological sections of gills, epithelial cells of primary and secondary lamellae stained positive with MHC class I antiserum, while single, scattered cells stained positive for MHC class II. In sections of spleen and head kidney, considerable numbers of cells positive for either MHC type were detected. Molecular weight shift in SDS-PAGE after deglycosylation of MHC class I alpha and class II beta confirmed the predicted glyco-protein character of the molecules. The majority of MHC II alpha was not glycosylated; only a small fraction of MHC II alpha was susceptible to deglycosylation. This suggests differential expression of the two stickleback MHC II alpha genes (Gaac-DAA, Gaac-DBA) only one of which (Gaac-DBA) has a site for N-linked glycosylation.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Sueros Inmunes/inmunología , Proteínas Recombinantes/inmunología , Smegmamorpha/inmunología , Animales , Anticuerpos/análisis , Anticuerpos/metabolismo , Western Blotting , Inmunohistoquímica/veterinaria , Datos de Secuencia Molecular , Conejos , Bazo/inmunologíaRESUMEN
Three-spined sticklebacks Gasterosteus aculeatus are frequent paratenic hosts of the nematode parasites Anguillicola crassus and Camallanus lacustris. As paratenic hosts, sticklebacks could spread infection by carrying high numbers of infective stages. In contrast, low infective ability of either parasite for the paratenic host could hinder the spread of infection. In the present study, G. aculeatus was, for the first time, infected under controlled laboratory conditions with defined doses of the parasites. Sticklebacks were exposed to 6, 12, 18 and 24 parasite larvae to determine the infective ability of the 2 nematode species. There were significantly higher infection rates for C. lacustris (18 to 49%) than for A. crassus (4 to 14%) at each exposure dose. In C. lacustris-infected sticklebacks, infection rates tended to be highest after exposure to 12 C. lacustris larvae and lowest after exposure to 24 parasites. In A. crassus-infected sticklebacks, no effect of parasite exposure dose on infection rates was observed. Immunity parameters such as respiratory burst activity and lymphocyte proliferation of head kidney leukocytes recorded 18 wk post exposure were not significantly affected by either parasite or exposure dose. Granulocyte:lymphocyte ratios were elevated only within the stickleback group showing the highest infection intensity of C. lacustris, i.e. to those exposed 18 parasites.
Asunto(s)
Enfermedades de los Peces/parasitología , Nematodos/patogenicidad , Infecciones por Nematodos/veterinaria , Smegmamorpha/parasitología , Animales , Copépodos/parasitología , Dracunculoidea/patogenicidad , Granulocitos , Linfocitos/sangre , Nematodos/aislamiento & purificación , Infecciones por Nematodos/parasitología , Estallido Respiratorio/inmunología , Espirúridos/patogenicidad , Infecciones por Spirurida/parasitología , Infecciones por Spirurida/veterinariaRESUMEN
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.
Asunto(s)
Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Inmunidad Innata/inmunología , Kinetoplastida , Infecciones Protozoarias en Animales , Análisis de Varianza , Animales , Anticuerpos/metabolismo , Carpas , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida/veterinaria , Citometría de Flujo/veterinaria , Sueros Inmunes/inmunología , Leucocitos/inmunología , Infecciones por Protozoos/inmunologíaRESUMEN
Leukocytes isolated from the head kidney and peripheral blood of 3-spined sticklebacks Gasterosteus aculeatus L. were analysed by means of flow cytometry during infection with the tapeworm Schistocephalus solidus (Müller, 1776). Although parasites increased their body weight continuously throughout the observation period (98 d), proportions of granulocytes increased in blood and head kidney only up to Day 63 post-infection (p.i.). Thereafter, declining proportions of granulocytes were observed in both organs. Thus the relative decrease in granulocyte number was not correlated to a decline in the parasitic load of the fish. To investigate a possible modulatory impact of S. solidus on granulocyte function, head kidney leukocytes were isolated at times before Day 63 p.i. and tested in vitro for their capacity to produce reactive oxygen species (ROS). Head kidney leukocytes from S. solidus-infected fish, analysed immediately after isolation (ex vivo, Day 40 p.i.), exhibited a higher ROS production when stimulated with phorbol myristate acetate (PMA), than leukocytes from naive, sham-treated control fish and fish that had resisted or cleared the infection (exposed but not infected). The latter showed an increased spontaneous ROS production that was not correlated to the numbers of granulocytes present in the head kidney isolates. In infected sticklebacks, spontaneous and PMA-induced ROS production was significantly correlated with numbers of granulocytes present in the head kidney isolates, suggesting that elevated ROS production was due to higher numbers of responding cells rather than an increased capacity of single cells. In vitro, after cultivation for 4 d in the presence of pokeweed mitogen (PWM) or extracts from S. solidus, head kidney leukocytes from control fish showed an increased ROS production and phagocytic activity compared with non-stimulated control cultures. In contrast, head kidney leukocytes from infected fish isolated on Days 48 and 44 p.i., failed to respond to S. solidus antigens in vitro. During S. solidus infection, granulocyte mobilisation resulted in elevated numbers of these cells in head kidneys, but the lack of an in vitro response to S. solidus antigens indicates an in vivo priming of granulocytes by the parasite. These observations may reflect the ability of S. solidus to impair the host's immune response once the parasite is developing in the body cavity of G. aculeatus.
Asunto(s)
Difilobotriosis/veterinaria , Diphyllobothrium/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Granulocitos/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Análisis de Varianza , Animales , Difilobotriosis/inmunología , Citometría de Flujo , Granulocitos/inmunología , Granulocitos/metabolismo , Riñón/citología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/inmunología , SmegmamorphaRESUMEN
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.
Asunto(s)
Carpas/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/parasitología , Kinetoplastida/inmunología , Neutrófilos/inmunología , Animales , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/veterinaria , Riñón/citología , Riñón/inmunología , Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Nitroazul de Tetrazolio/metabolismo , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Estadísticas no Paramétricas , omega-N-Metilarginina/farmacologíaRESUMEN
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.
Asunto(s)
Carpas/inmunología , Enfermedades de los Peces/parasitología , Kinetoplastida/inmunología , Leucocitos Mononucleares/inmunología , Óxido Nítrico/biosíntesis , Infecciones Protozoarias en Animales , Espermina/análogos & derivados , Animales , Inhibidores Enzimáticos/farmacología , Enfermedades de los Peces/inmunología , Citometría de Flujo/veterinaria , Kinetoplastida/crecimiento & desarrollo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/parasitología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/parasitología , Óxido Nítrico/inmunología , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Fagocitosis/inmunología , Infecciones por Protozoos/inmunología , Infecciones por Protozoos/parasitología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , S-Nitrosoglutatión/farmacología , Espermina/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
Haemolytic substances produced by ichthyotoxic algae often are unknown in molecular structure or specific mechanism of toxicity. Detection and quantification of such substances are dependent on bioassays, using markers that are sensitive for haemolytic impairment and generation of a recordable response. The erythrocyte lysis assay (ELA) represents an advantageous bioassay in this respect, because the lytic response can be measured photometrically by the amount of released haemoglobin. The aim of the present study was to establish an improved assay based on the ELA principle, for sensitive determination of haemolytic substances of microalgae and for high sample throughput. For this purpose we adapted the ELA to a 96-well microtitre plate format, which significantly reduced the sample volumes and allowed rapid processing of samples. Further improvement was achieved by measuring absorption of lysed erythrocytes at 414 nm, which significantly increased the sensitivity of the ELA compared to the measurements at 540 nm that are usually applied in this type of assay. Using carp (Cyprinus carpio) erythrocytes it was possible to detect haemolysis induced by 4 microg ml(-1) of saponin and as little as two haemolytic Alexandrium tamarense cells. It is suggested that this improved ELA in microtitre plates be used as a low-cost monitoring tool for detection and analysis of potential harmful algae. Furthermore, this ELA can be useful as a sensitive screening system for substances of pharmacological interest, e.g. selectively acting cytolytic antibiotics.
Asunto(s)
Dinoflagelados , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Animales , Bioensayo/métodos , Carpas , Eritrocitos/patología , Fotometría , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Proliferation of rainbow trout peripheral blood leucocytes in vitro is usually assessed by measuring incorporated tritiated thymidine. In this report we monitored the in vitro proliferative response to the mitogen Concanavalin A (Con A) by means of flow cytometry (FCM) and 3H-thymidine incorporation. When analysed by FCM, blood leucocytes displayed two main cell populations with distinct forward and side scatter (FSC/SSC) characteristics: lymphocytes with low FSC/SSC values and non-lymphoid leucocytes (NLL) with increased FSC/SSC values. The nature of these cell types were confirmed by microscopy. Interestingly, the FSC/SSC pattern of lymphocytes remained unchanged after in vitro stimulation with Con A, whereas cells from the NLL population showed a marked shift towards increased FSC values. In stimulated cultures, the increase of FSC values of the NLL population significantly correlated with contemporarily measured 3H-thymidine incorporation (r = 0.7, P < 0.001). The mitogenic response of blood leucocytes originating from different individual fish varied over wide ranges. It was found to be related to the numbers of NLL present in the leucocyte sample. The present results show that qualitative and quantitative FCM analysis of morphological parameters (FSC/SSC) of blood leucocytes makes it possible to discriminate between leucocyte populations of the rainbow trout and to monitor cell proliferation experiments.
Asunto(s)
Leucocitos/efectos de los fármacos , Mitógenos/farmacología , Oncorhynchus mykiss/inmunología , Animales , División Celular , Células Cultivadas , Citometría de Flujo/veterinaria , Leucocitos/inmunología , Microscopía de Contraste de Fase/veterinaria , Oncorhynchus mykiss/sangreRESUMEN
An in vitro culture system was developed for Trypanoplasma borreli, a pathogenic flagellate from the blood of European cyprinids. Trypanoplasms multiplied rapidly in a mixture of Hanks' balanced salt solution (HBSS, 45%), L15 (22.5%), Earle's minimum essential medium (MEM, 22.5%) and 10% distilled water, which was supplemented with 5 to 10% heat-inactivated pooled carp serum. In medium supplemented with fetal bovine serum, multiplication of T. borreli seemed to be inhibited. Cultures initiated with less than 100 000 T. borreli per ml culture medium did not survive, and a substantial multiplication of trypanoplasms was found at inocula beginning with 630 000 flagellates ml(-1). Trypanoplasms multiplied at 15, 20 and 25 degrees C. In cultures incubated at 4 degrees C the trypanoplasms remained viable but the number of flagellates did not increase. Trypanoplasms from in vitro cultures retained their infectivity for carp for at least 90 d (5 passages). The trypanoplasms survived in culture over a period of up to 5 mo (10 passages). The established culture system allows the propagation of high numbers of fish-infective trypanoplasms, which are required to study parasite-host relationships in carp.
Asunto(s)
Carpas/parasitología , Kinetoplastida/aislamiento & purificación , Parasitología/métodos , Animales , Femenino , Citometría de Flujo/veterinaria , Carpa Dorada/parasitologíaRESUMEN
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.