Kurt Buchmann

Teleost skin, an ancient mucosal surface that elicits gut-like immune responses

Skin homeostasis is critical to preserve animal integrity. Although the skin of most vertebrates is known to contain a skin-associated lymphoid tissue (SALT), very little is known about skin B-cell responses as well as their evolutionary origins. Teleost fish represent the most ancient bony vertebrates containing a SALT. Due to its lack of keratinization, teleost skin possesses living epithelial cells in direct contact with the water medium. Interestingly, teleost SALT structurally resembles that of the gut-associated lymphoid tissue, and it possesses a diverse microbiota. Thus, we hypothesized that, because teleost SALT and gut-associated lymphoid tissue have probably been subjected to similar evolutionary selective forces, their B-cell responses would be analogous. Confirming this hypothesis, we show that IgT, a teleost immunoglobulin specialized in gut immunity, plays the prevailing role in skin mucosal immunity. We found that IgT+ B cells represent the major B-cell subset in the skin epidermis and that IgT is mainly present in polymeric form in the skin mucus. Critically, we found that the majority of the skin microbiota are coated with IgT. Moreover, IgT responses against a skin parasite were mainly limited to the skin whereas IgM responses were almost exclusively detected in the serum. Strikingly, we found that the teleost skin mucosa showed key features of mammalian mucosal surfaces exhibiting a mucosa-associated lymphoid tissue. Thus, from an evolutionary viewpoint, our findings suggest that, regardless of their phylogenetic origin and tissue localization, the chief immunoglobulins of all mucosa-associated lymphoid tissue operate under the guidance of primordially conserved principles.

Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals

Host responses against invading pathogens are basic physiological reactions of all living organisms. Since the appearance of the first eukaryotic cells, a series of defense mechanisms have evolved in order to secure cellular integrity, homeostasis, and survival of the host. Invertebrates, ranging from protozoans to metazoans, possess cellular receptors, which bind to foreign elements and differentiate self from non-self. This ability is in multicellular animals associated with presence of phagocytes, bearing different names (amebocytes, hemocytes, coelomocytes) in various groups including animal sponges, worms, cnidarians, mollusks, crustaceans, chelicerates, insects, and echinoderms (sea stars and urchins). Basically, these cells have a macrophage-like appearance and function and the repair and/or fight functions associated with these cells are prominent even at the earliest evolutionary stage. The cells possess pathogen recognition receptors recognizing pathogen-associated molecular patterns, which are well-conserved molecular structures expressed by various pathogens (virus, bacteria, fungi, protozoans, helminths). Scavenger receptors, Toll-like receptors, and Nod-like receptors (NLRs) are prominent representatives within this group of host receptors. Following receptor–ligand binding, signal transduction initiates a complex cascade of cellular reactions, which lead to production of one or more of a wide array of effector molecules. Cytokines take part in this orchestration of responses even in lower invertebrates, which eventually may result in elimination or inactivation of the intruder. Important innate effector molecules are oxygen and nitrogen species, antimicrobial peptides, lectins, fibrinogen-related peptides, leucine rich repeats (LRRs), pentraxins, and complement-related proteins. Echinoderms represent the most developed invertebrates and the bridge leading to the primitive chordates, cephalochordates, and urochordates, in which many autologous genes and functions from their ancestors can be found. They exhibit numerous variants of innate recognition and effector molecules, which allow fast and innate responses toward diverse pathogens despite lack of adaptive responses. The primitive vertebrates (agnathans also termed jawless fish) were the first to supplement innate responses with adaptive elements. Thus hagfish and lampreys use LRRs as variable lymphocyte receptors, whereas higher vertebrates [cartilaginous and bony fishes (jawed fish), amphibians, reptiles, birds, and mammals] developed the major histocompatibility complex, T-cell receptors, and B-cell receptors (immunoglobulins) as additional adaptive weaponry to assist innate responses. Extensive cytokine networks are recognized in fish, but related signal molecules can be traced among invertebrates. The high specificity, antibody maturation, immunological memory, and secondary responses of adaptive immunity were so successful that it allowed higher vertebrates to reduce the number of variants of the innate molecules originating from both invertebrates and lower vertebrates. Nonetheless, vertebrates combine the two arms in an intricate inter-dependent network. Organisms at all developmental stages have, in order to survive, applied available genes and functions of which some may have been lost or may have changed function through evolution. The molecular mechanisms involved in evolution of immune molecules, might apart from simple base substitutions be as diverse as gene duplication, deletions, alternative splicing, gene recombination, domain shuffling, retrotransposition, and gene conversion. Further, variable regulation of gene expression may have played a role.

Innate immune response in rainbow trout (Oncorhynchus mykiss) against primary and secondary infections with Yersinia ruckeri O1

Response mechanisms in teleosts against bacterial pathogens have been widely studied following injection procedures applying preparations of killed bacteria. In contrast, investigations on immune reactions in fish which have survived a primary infection and subsequently have been challenged are few or lacking. However, knowledge on these factors during infection and re-infection could provide the basis for development of improved vaccines. The innate immune response in rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri O1 has been studied following a primary intra-peritoneal injection with 5 x 10(5) CFU Y. ruckeri, and after bacterial clearance a secondary infection 35 days later. The number of pathogens in the liver was measured with a Y. ruckeri specific 16S ribosomal RNA quantitative real-time RT-PCR (q-PCR) during the course of infection. The bacterial counts peaked on day 3 during the primary infection and were significantly lower during the re-infection. Re-challenged fish showed a highly increased survival when compared to the naïve fish receiving a primary infection indicating development of adaptive immunity in the fish against this bacterial pathogen. We investigated the gene expression of innate immune factors in the liver during infections in order to elucidate molecules involved in survival of hosts before adaptive immunity was mounted. Transcription of mRNA was measured in liver samples taken 8 h, 1, 3, 7, 14 and 28 d post-infection using q-PCR. The investigation focused on genes encoding toll-like receptor 5 (TLR5), the pro-inflammatory cytokines IL-1beta, IL-6 and TNF-alpha, the acute phase proteins (APPs) serum amyloid protein a (SAA), trout C polysaccharide binding protein, a CRP/SAP like pentraxin, precerebellin, transferrin, hepcidin and finally the complement factors C3, C5 and factor B. Infection elicited significantly increased gene expression of all the cytokines (IL-6 > 1000-fold), some acute phase proteins (SAA > 3000-fold) and down-regulation of complement factors (C3, C5 and factor B). SAA expression was significantly earlier activated during the re-infection when compared to the primary infection. The pattern of gene activation suggested that the innate response was based on pathogen binding to toll-like receptors, production of cytokines and subsequent release of APPs. In general, both the innate immune response and the amount of Y. ruckeri measured in the liver during the re-infection was much lower compared to the first infection, probably reflecting development of adaptive immunity.

Bath vaccination of rainbow trout (Oncorhynchus mykiss Walbaum) against Yersinia ruckeri : Effects of temperature on protection and gene expression

Protection of rainbow trout fry following bath vaccination with a bacterin of Y. ruckeri O1, the bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15 and 25 degrees C. Rainbow trout fry were acclimatised for 8 weeks at the three temperatures before vaccination. They were subsequently challenged with Y. ruckeri 4 and 8 weeks post-vaccination which demonstrated a significant protection of vaccinated fish kept at 15 degrees C. No protective effect of vaccination in rainbow trout reared at 5 and 25 degrees C could be recorded. Spleen tissue was sampled from vaccinated and control fish at 0, 8, 24 and 72 h post-vaccination in order to analyse gene transcript profiles using quantitative real-time RT-PCR (q-PCR). Gene expression in fish vaccinated at 15 degrees C (the protected fish) was up-regulated with regard to the pro-inflammatory cytokines IFN-gamma, TNF-alpha, IL-6 and the anti-inflammatory cytokines IL-10 and TGF-beta, the cell receptors TcR, CD8alpha, CD4, C5aR and the teleost specific immunoglobulin IgT. Passive immunisation using transfer of plasma from vaccinated fish to naïve fish conferred no protection. This indicates that humoral factors such as Ig and complement are less important in the protection induced by bath vaccination. Expression of cellular factors such as CD8alpha was significantly increased in the protected trout and this suggests that cellular factors including cytotoxic T-cells could play a role in immunity against Y. ruckeri.

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies.

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.

Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis

Recent studies have shown that fish are able to mount protective immune responses against various parasites. One of the best characterized parasite-host system in this context is the ciliate Ichthyophthirius multifiliis (Ich) parasitizing a range of freshwater fishes. Both specific and non-specific host defence mechanisms are responsible for the protection of fish against challenge infections with this ciliate. The specific humoral components comprise at least specific antibodies. The non-specific humoral elements included are the alternative complement pathway and probably lectins. Cellular factors involved in the specific response are B-cells and putative T-cells. The non-specific effector cells recognized are various leukocytes. In addition, goblet-cells and mast cells (EGC-cells) may have a function. The NCC-cell (suggested analogue to NK-cells in mammals) seems to play a role in the non-specific response. This well documented protective response in freshwater fishes against Ich has urged the development of anti-parasitic vaccines. Indeed, such products based on formalin killed parasites have been developed and found to offer the vaccinated host a satisfactory protection. However, the collection of parasites for vaccine production is extremely laborious. It involves keeping infected fish due to the fact that in vitro propagation of the parasite is still insufficiently developed. Gaining knowledge of amino acid sequences and its encoding DNA-sequences for the protective antigens (i-antigens) in the parasite was a major breakthrough. That achievement made it possible to produce a recombinant protein in E. coli and preliminary results indicated a certain protection of fish vaccinated with this product. Recent work has shown that the free-living and easily cultivated ciliate Tetrahymena can be transformed and express the i-antigen. This path seems to be promising for future development of vaccines against Ich. A novel approach in fish is the development of DNA-vaccines. Successful DNA-vaccination trials have been conducted in fish against viral infections and the technology also makes it possible to develop a DNA-vaccine against Ich. Other approaches to immuno-protection against Ich have been the use of heterologous vaccines. Thus, both bath and injection vaccination using live or killed (un-transformed) Tetrahymena has been reported to offer treated fish a certain level of protection. Such protection could be explained by non-specific reactions and the efficacy and duration of this vaccination type should be further evaluated.

Health of farmed fish: its relation to fish welfare and its utility as welfare indicator

This brief review focuses on health and biological function as cornerstones of fish welfare. From the function-based point of view, good welfare is reflected in the ability of the animal to cope with infectious and non-infectious stressors, thereby maintaining homeostasis and good health, whereas stressful husbandry conditions and protracted suffering will lead to the loss of the coping ability and, thus, to impaired health. In the first part of the review, the physiological processes through which stressful husbandry conditions modulate health of farmed fish are examined. If fish are subjected to unfavourable husbandry conditions, the resulting disruption of internal homeostasis necessitates energy-demanding physiological adjustments (allostasis/acclimation). The ensuing energy drain leads to trade-offs with other energy-demanding processes such as the functioning of the primary epithelial barriers (gut, skin, gills) and the immune system. Understanding of the relation between husbandry conditions, allostatic responses and fish health provides the basis for the second theme developed in this review, the potential use of biological function and health parameters as operational welfare indicators (OWIs). Advantages of function- and health-related parameters are that they are relatively straightforward to recognize and to measure and are routinely monitored in most aquaculture units, thereby providing feasible tools to assess fish welfare under practical farming conditions. As the efforts to improve fish welfare and environmental sustainability lead to increasingly diverse solutions, in particular integrated production, it is imperative that we have objective OWIs to compare with other production forms, such as high-density aquaculture. However, to receive the necessary acceptance for legislation, more robust scientific backing of the health- and function-related OWIs is urgently needed.

Treatment of trichodiniasis in eel Anguilla / anguilla reared in recirculation systems in Denmark: alternatives to formaldehyde

Treatment of trichodiniasis in recirculation eel farms using formalin (37%) has been found insufficient. We have screened 30 different chemical substances for efficacy against natural infections with trichodinids (Trichodina jadranica) on skin and gills of eels. The following substances showed high parasiticidal effect: acriflavin (25 ppm), bithionol (0.1 ppm), chloramine T (50 ppm), Detarox AP® (45 ppm), malachite green (1 ppm), raw garlic (200 ppm), potassium permanganate (20 ppm) and Virkon PF® vet. (20 ppm). Preliminary screening revealed that the anthelmintic, bithionol, and the decomposable disinfectants Detarox AP and Virkon PF® vet. were potential therapeutics and these were tested on infected eel stocks in recirculation production units. The following treatments showed a high parasiticidal effect: bithionol at 0.1 ppm in a recirculation system including the biofilters; Detarox AP® (45 ppm) in recirculation systems with the biofilters temporarily isolated for 1 h and Virkon PF® vet. (40 ppm; 25 ppm at start supplemented with 15 ppm 1 h later) in rearing tanks temporarily isolated from recirculation systems for 3 h.

PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss).

Host immune responses elicited by invading pathogens depend on recognition of the pathogen by specific receptors present on phagocytic cells. However, the reactions to viral, bacterial, parasitic and fungal pathogens vary according to the pathogen-associated molecular patterns (PAMPs) on the surface of the invader. Phagocytic cells are known to initiate a respiratory burst following an exposure to the pathogen, but the underlying and associated specific elements are poorly elucidated in fish. The present study describes the differential response of head kidney leukocytes from rainbow trout (Oncorhynchus mykiss) to different PAMPs mimicking viral (poly I:C), bacterial (flagellin and LPS) and fungal infections (zymosan and β-glucan). Transcript of cytokines related to inflammation (IL-1β, IL-6, IL-10 and TNF-α) was highly up-regulated following LPS exposure whereas flagellin or poly I:C induced merely moderate reactions. In contrast, IFN-γ expression was significantly higher in the poly I:C stimulated group compared to the LPS group. When head kidney cells were exposed to zymosan or β-glucan, genes encoding IL-1β, TNF-α, IL-6 and IL-10 became up-regulated. Their level of up-regulation was comparable to LPS but the kinetics differed. In particular, TNF-α induction was considerably slower when stimulated with zymosan or β-glucan. The gene encoding the COX-2 enzyme, a central element during initiation of inflammatory reactions, was significantly higher in stimulated cells although a depressing effect of high concentrations of LPS and zymosan became evident after 4h exposure. This study suggests that rainbow trout leukocytes respond differently to viral, bacterial and fungal PAMPs, which may reflect activation of specific signaling cascades eventually leading to activation of different immune effector molecules.

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non-lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.