The myxozoan parasite Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD)-a disease of salmonid fishes, notably of the commercially farmed rainbow trout Oncorhynchus mykiss. Both wild and farmed salmonids are threatened by this virulent/deadly disease, a chronic immunopathology characterized by massive lymphocyte proliferation and hyperplasia, which manifests as swollen kidneys in susceptible hosts. Studying the immune response towards the parasite helps us understand the causes and consequences of PKD. While examining the B cell population during a seasonal outbreak of PKD, we unexpectedly detected the B cell marker immunoglobulin M (IgM) on red blood cells (RBCs) of infected farmed rainbow trout. Here, we studied the nature of this IgM and this IgM+ cell population. We verified the presence of surface IgM via parallel approaches: flow cytometry, microscopy, and mass spectrometry. The levels of surface IgM (allowing complete resolution of IgM- RBCs from IgM+ RBCs) and frequency of IgM+ RBCs (with up to 99% of RBCs being positive) have not been described before in healthy fishes nor those suffering from disease. To assess the influence of the disease on these cells, we profiled the transcriptomes of teleost RBCs in health and disease. Compared to RBCs originating from healthy fish, PKD fundamentally altered RBCs in their metabolism, adhesion, and innate immune response to inflammation. In summary, RBCs play a larger role in host immunity than previously appreciated. Specifically, our findings indicate that the nucleated RBCs of rainbow trout interact with host IgM and contribute to the immune response in PKD.
- MeSH
- B-lymfocyty MeSH
- erytrocyty MeSH
- imunoglobulin M MeSH
- nemoci ledvin * MeSH
- Oncorhynchus mykiss * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest μm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.
The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.
Myxozoans are a diverse group of cnidarian parasites, including important pathogens in different aquaculture species, without effective legalized treatments for fish destined for human consumption. We tested the effect of natural feed additives on immune parameters of common carp and in the course of a controlled laboratory infection with the myxozoan Sphaerospora molnari. Carp were fed a base diet enriched with 0.5% curcumin or 0.12% of a multi-strain yeast fraction, before intraperitoneal injection with blood stages of S. molnari. We demonstrate the impact of these treatments on respiratory burst, phagocytosis, nitric oxide production, adaptive IgM+ B cell responses, S. molnari-specific antibody titers, and on parasite numbers. Both experimental diets enriched B cell populations prior to infection and postponed initial parasite proliferation in the blood. Curcumin-fed fish showed a decrease in reactive oxygen species, nitric oxide production and B cell density at late-stage infection, likely due to its anti-inflammatory properties, favoring parasite propagation. In contrast, multi-strain yeast fraction (MsYF)-fed fish harbored the highest S. molnari-specific antibody titer, in combination with the overall lowest parasite numbers. The results demonstrate that yeast products can be highly beneficial for the outcome of myxozoan infections and could be used as effective feed additives in aquaculture.
- Publikační typ
- časopisecké články MeSH
AIMS: As the most abundant cell population in the blood, erythrocytes represent an attractive source of nutrients and a protective niche to a number of pathogens. Previously, we observed the attachment of the myxozoan parasite Sphaerospora molnari to erythrocytes of its host, common carp (Cyprinus carpio), raising a number of questions about the nature of this interaction. METHODS AND RESULTS: We elucidated the impact of S molnari on the number of erythrocytes in healthy and immunocompromised fish, over a period of 6 weeks. While we observed only a mild decrease in RBC numbers in healthy individuals, we witnessed gradual and finally severe haemolytic anaemia in immunosuppressed fish. Accompanying this overt loss was increased erythropoiesis as represented by an increase of erythroblasts in the blood. In vitro, we demonstrated the uptake of host proteins from CFSE-labelled erythrocytes, ultimately inducing death of host RBCs, likely for nutrient gain of the parasite. Nevertheless, the results do not exclude a possible role of erythrocyte-derived proteins in immune evasion. CONCLUSION: Overall, the obtained data provide first evidence for the previously unknown appetite of myxozoan parasites for host erythrocytes and create an important framework for future investigations into the molecular mechanisms underlining this interaction.
- MeSH
- erytrocyty parazitologie MeSH
- erytropoéza fyziologie MeSH
- fylogeneze MeSH
- hemolytické anemie parazitologie MeSH
- kapři parazitologie MeSH
- Myxozoa fyziologie MeSH
- nemoci ryb parazitologie MeSH
- stravovací zvyklosti fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout's physiology, especially on the immune system.
- MeSH
- glukosa metabolismus MeSH
- hemoglobiny analýza MeSH
- hydrokortison krev MeSH
- imunitní systém imunologie MeSH
- nahuštění v prostoru * MeSH
- Oncorhynchus mykiss genetika imunologie MeSH
- reakce na tepelný šok * MeSH
- rybí proteiny genetika metabolismus MeSH
- slezina imunologie metabolismus MeSH
- stanovení celkové genové exprese MeSH
- transkriptom * MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year. RESULTS: We have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (< 40 hours), an initial covert infection period with a moderate proinflammatory response for about 1-2 weeks, followed by a period of parasite multiplication in the blood which peaked at 28 days post-infection (dpi) and was associated with a massive lymphocyte response. Our data further revealed a switch to a massive anti-inflammatory response (up to 1456-fold expression of il-10), a strong increase in the expression of IgM transcripts and increased number of IgM+ B lymphocytes, which produce specific antibodies for the elimination of most of the parasites from the fish at 35 dpi. However, despite the presence of these antibodies, S. molnari invades the liver 42 dpi, where an increase in parasite cell number and indistinguishable outer cell membranes are indicative of effective exploitation and disguise mechanisms. From 49 dpi onwards, the acute infection changes to a chronic one, with low parasite numbers remaining in the fish. CONCLUSIONS: To our knowledge, this is the first time myxozoan early development and immune modulation mechanisms have been analyzed along with innate and adaptive immune responses of its fish host, in a controlled laboratory system. Our study adds important information on host-parasite interaction and co-evolutionary adaptation of early metazoans (Cnidaria) with basic vertebrate (fish) immune systems and the evolution of host adaptation and parasite immune evasion strategies.
- MeSH
- buněčná imunita MeSH
- cytokiny metabolismus MeSH
- hlavová ledvina metabolismus MeSH
- humorální imunita MeSH
- interakce hostitele a parazita MeSH
- kapři imunologie parazitologie MeSH
- modely nemocí na zvířatech MeSH
- Myxozoa růst a vývoj imunologie MeSH
- nemoci ryb imunologie parazitologie MeSH
- parazitární nemoci u zvířat imunologie parazitologie MeSH
- spory MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.
