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.
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 (
In the blood, thrombocytes and all leukocyte types showed a substantial increase over time in response to S. molnari infection in carp (Fig. 2) with a maximum increase in lymphocytes (up to 13-fold), especially B cells and monocytes (up to 13-fold), followed by neutrophils (up to 10-fold) and thrombocytes (up to 3-fold), in individual fish. Thrombocyte numbers were higher in infected fish on all days except 1, 2, 14 and 42 dpi (Fig. 2). From 35 dpi onwards, we observed an increase in the number of thrombocytes, with the exception of 42 dpi, when S. molnari resided in the liver and fewer thrombocytes were detected in the blood. This decrease at 42 dpi and during the occurrence of LS was also noticed in other immune cells such as lymphocytes and monocytes, while less obvious in neutrophils. Neutrophil and monocyte numbers (Fig. 2) increased from 14 dpi onwards and remained elevated until the end of the experiment. Neutrophils first peaked at 14 dpi and then reached their maximum at 35 dpi, when S. molnari blood stages are reduced in the blood. Monocytes also first increased 14 dpi and were considerably elevated at 21, 28, 35 and 56 dpi, while showing a decrease towards the end of the experiment (63 dpi). As the most obvious cellular response to S. molnari infection, the number of lymphocytes in infected fish increased massively, from 21 dpi, when S. molnari appeared for the first time in the blood. Numbers thereafter increased considerably, with a significant difference to the control group at all time points except for 42 dpi, and a slight decrease noticeable at 63 dpi, similar to monocytes and coincident with the initiation of a chronic low-level infection by S. molnari at 49 dpi.
Meanwhile, il-6 and tnfα were upregulated throughout the study indicating a prolonged activation of innate immunity. The increase of tnfα has been reported in other models including carp infected with Trypanosoma borreli  and in all examined organs of fish infected by E. leei . Notably, tnfα does not only play the classical role by stimulating the production of a number of genes associated with inflammation, enhancing the phagocytic activity of leukocytes, regulating homing, proliferation and migration , but it also exhibits lectin-like activity towards parasites such as Trypanosoma brucei . However, the effect of tnfα is not always beneficial, as the excessive production of tnfα was shown to be associated with the development of lesions and loss of barrier function during enteromyxosis in turbot . il-6 is known for its pleitropic effect playing a pivotal role during the transition from innate to acquired immunity. In fish, it has been reported to promote macrophage growth and production of antimicrobial proteins , while at the same time supporting antibody production [88, 89]. Possibly, in the S. molnari infection it might be related to the attraction of monocytes, secretion of IgM from 21 dpi onward and the resolution of the inflammation in the final stages. Involvement of il-6 has also been described in E. leei infected gilthead sea bream  and C. shasta infected Chinook salmon .
From the selected cytokines, the anti-inflammatory il-10 underwent the strongest upregulation, reaching up to 1456-fold increase in expression compared to control fish, at 56 dpi. As carp has two paralogues of il-10 which have almost identical gene structure, synteny, protein sequence and exert identical biological activities in vitro, it is worth noting that the primers used in the present study were targeting the il-10b paralogue, which is preferentially upregulated upon infection . Traditionally, il-10 is not only associated with the control of excessive immune responses but in cell cultures obtained from T. borreli-infected carp, il-10 promotes the proliferation, differentiation and antibody secretion by the IgM+ B cells . Importantly, a growing evidence from other disease models suggest that the protective function of il-10 can be exploited by pathogens . In infection with other Myxozoa, high levels of il-10 were induced by C. shasta , T. bryosalmonae  and also in the intestine, but not in head kidney of gilthead sea bream infected E. leei . These results point to il-10 functioning as a surrogate of myxozoan infections and question its role in host-pathogen interaction. Possibly, the expression of il-10 can be explained by two scenarios. In the traditional understanding, il-10 is induced by the immune system itself in an effort to minimize the collateral damage caused by neutrophils and monocytes fighting the growing number of S. molnari. Alternatively, S. molnari uses an unknown mechanism to induce il-10 as an immunomodulation strategy to deactivate the effector capacities of the immune system. The present data does not exclude any of these scenarios and future studies will investigate whether high concentrations of il-10 are a cause or a consequence of high pathogen burdens.
To elucidate the nature of the B cell responses, we investigated the presence of specific antibodies to S. molnari by SDS PAGE of parasite antigens and subsequent western blotting with sera of infected fish, which identified a single protein of 16 kDa. While the identity of this protein remains to be determined, it is noteworthy that an antigenic protein of the same size (and an additional 165 kDa protein) was characterized in E. leei, using a similar approach . The two proteins could well correspond to common antigens shared among myxozoans. Estensoro et al.  showed the E. leei antigen to be a glycoprotein, and suggested a minicollagen; however, minicollagens would unlikely be expressed in proliferative stages of S. molnari as they are essential components of polar capsules and cnidarian stinging cells [100, 101], expressed during spore formation. Nevertheless, while the western blot clearly demonstrated the acquisition of specific immunity of carp against S. molnari, the low titer of specific antibodies in combination with an extremely high numbers of circulating B cells (up to 9-fold increase in number) indirectly confirms the suspicion that S. molnari, like other myxozoan parasites, manipulates B cells responses.
Our study demonstrates an activation of innate and adaptive humoral immune responses of common carp to the myxozoan S. molnari, during early stage of infection and parasite proliferation. We show that myxozoans use important immunomodulatory and immune evasion strategies such as intracellular disguise, motility, polyclonal activation of B cell responses and skewing of host response to an anti-inflammatory phenotype, in order to be able to successfully proliferate in their fish hosts and produce infective spore stages to continue their life-cycle. We believe that the present comprehensive characterization of host and parasite interactions between S. molnari and common carp represents a solid base for further research investigating newly raised questions in our in vivo model, with a focus on specific immune responses and universal myxozoan antigenic proteins that could in the future be used for targeted therapies and potential vaccine design.
To gain insights into the number and proportion of B lymphocytes from 7 dpi onwards and throughout the infection we adapted a protocol for the flow cytometric analysis of the full blood described previously . Briefly, 2 µl of blood of control and infected fish were washed with cold RPMI and stained for 20 min with a monoclonal antibody recognizing the heavy chain of carp IgM (1µg/ml) (Aquatic Diagnostics Ltd, Stirling, UK), followed by staining with goat-anti-mouse IgG Alexa Fluor 488 (2 µg/ml; Thermo Fisher Scientific, Pardubice, Czech Republic). The samples were washed twice and resuspended in 200 µl of RPMI. The proportion and total number of IgM+ B cells were determined using BD FACSCanto II (BD Biosciences, Prague, Czech Republic). Each sample was acquired for 30 s with a flow rate of 60 µl/min.
Transcriptomic data obtained from a mixture of white blood cells and S. molnari blood stages was mined for IgM heavy chain sequences of C. carpio. Two isoforms were found, one with a transmembrane domain (on B cell/plasma cell surface; GenBank: MH352353) and one with a secretory tail (secreted form; GenBank: MH352354). We developed TaqMan qPCR assays to differentiate between the expression of the secretory tetramer (IgMsec) and the membrane-bound monomer (IgMmem) (Additional file 1: Table S1). IgM expression was determined in the head kidney of experimental and control fish, after extraction of total RNA from the RNAlater-fixed samples using the NucleoSpin RNA (Macherey-Nagel, Düren, Germany) and cDNA preparation with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics). Both RNA as well as cDNA quality and quantity was determined by NanoDrop measurement. qPCRs were performed on samples adjusted to 100 ng/µl cDNA concentrations and relative to carp β-actin (Additional file 1: Table S1), using the same quality and compatibility measures as above. 2b1af7f3a8