The immune response of carp to blood flagellates : a model for studies on disease resistance and stress

<em></em>To date, aquaculture accounts for 25% of the total world supply of (shell)fish for human consumption, a relative contribution that is expected to increase with time. The increased global demand for (shell)fish has lead to a further intensification of aquaculture with the inevitable result that fish become disposed to stress and diseases. An important factor leading to this predisposition is stress induced by aquaculture practices such as crowding, transport, handling and impaired water quality. The World Health Organisation seeks to actively stimulate prophylactic measures such as vaccination, genetic selection and the use of immunomodulation by feed additives to prevent future disease outbreaks in aquaculture. Imperative for these approaches are <em>in vivo</em> infection models that allow reliable, reproducible challenge experiments to monitor efficacy of new treatments. <em>Trypanoplasma borreli</em> and <em>Trypanosoma carassii</em> are both protozoan kinetoplastid extracellular blood parasites of fish. The Kinetoplastida contain a number of parasites of major importance to man, <em>e.g.</em><em>Trypanosoma brucei</em> (sleeping sickness), <em>Leishmania</em> spp. (leishmaniasis), <em>Trypanosoma cruzi</em> (Chagas' disease). Both <em>T. borreli</em> and <em>T. carassii</em> are transmitted by blood-sucking leeches and infect cyprinids, the dominating species in freshwater aquaculture. Infection of carp with these parasites is an excellent model for comparative studies on host-parasite interactions with clear relevance to the problems faced by present day aquaculture ( <strong>chapter one</strong> ).Genetic selection for disease resistance can provide a major contribution to prophylaxis. One route to identify gene regions that determine susceptibility of fish to pathogens is the candidate gene approach. This approach is making use of known types of responses that have been proven important in the development of innate and acquired protective immunity. In <strong>chapter two</strong> the sequence of a candidate gene: the carp natural resistance-associated macrophage protein (NRAMP) is described. This protein is a putative metal transporter. Metals such as iron are essential nutrients for pathogens. Therefore, reducing iron availability can be an important part of the host defence strategy. Moreover, iron acts as a catalyst in the production of molecules such as hydroxyl radicals (OH<FONT FACE="Symbol">·</font>), which act as toxicants in the defence against intracellular pathogens. In <strong>chapter three</strong> the sequence of a second candidate gene: carp inducible nitric oxide synthase (iNOS) is described. Not only oxygen but also nitrogen radicals, produced by phagocytes, can act as toxicants, forming an important innate defence mechanism against pathogens. <em>Trypanoplasma borreli</em> or bacterial cell wall products induced iNOS expression in carp head kidney phagocytes leading to the production of high concentrations of the nitric oxide (NO) radical. The NO produced <em>in vitro</em> by head kidney phagocytes was cytostatic to the parasite.Carp challenged <em>in vivo</em> with <em>T. borreli</em> produce high amounts of NO ( <strong>chapter four</strong> ). The production of toxic molecules such as NO is potentially dangerous. In fact NO overproduction can lead to tissue damage in the host. Indeed, <em>in vivo</em> inhibition of NO production led to a higher rather than a lower survival of infected carp. A possible explanation for the harmful effect of NO <em>in vivo</em> could be the observation that, at least <em>in vitro</em> , NO can inhibit the proliferation of carp lymphocytes. Interestingly, in clear contrast with the effect of <em>T. borreli</em> , <em>T. carassii</em> did not induce production of NO.Lymphocytes are much more susceptible to the cytostatic effect of NO than phagocytes, which are mainly macrophages and neutrophilic granulocytes ( <strong>chapter five</strong> ). This difference could be ascribed to the fact that lymphocytes had lower levels of the most important cellular antioxidant glutathione (GSH). Furthermore, lymphocytes had lower levels of key enzymes involved in the maintenance of GSH compared to phagocytes.In <strong>chapter six</strong> we describe two sequences for carp tumour necrosis factor (TNF)<FONT FACE="Symbol">a</font>, which can be considered a third candidate gene for resistance to diseases of fish. Indeed, a polymorphism in carp TNF<FONT FACE="Symbol">a</font>2 could be associated with trypanotolerance. TNF<FONT FACE="Symbol">a</font>is a cytokine produced mainly by phagocytes in response to inflammation, infection and other physiological challenges. <em>In vitro</em> , <em>T. borreli</em> could induce expression of TNF<FONT FACE="Symbol">a</font>, which mediated the production of NO by phagocytes and the proliferation of leukocytes.To study the exact role of phagocytes in the immune defence against <em>T. borreli</em> , we applied a technique to deplete carp of macrophages, <em>in vivo</em> ( <strong>chapter seven</strong> ). These animals became more susceptible to opportunistic bacterial infections. When infected with blood flagellate parasites, however, there was a moderate increase in parasitaemia only, demonstrating that macrophages do not play a major role in the resistance against <em>T. borreli</em> or <em>T. carassii</em> . Carp surviving an infection with <em>T. borreli</em> are resistant to re-infection for more than 12 months. This acquired resistance was not abrogated when the animals were depleted of macrophages.The major immunogenic molecules of <em>T. borreli</em> are proteins (probably membrane glycoproteins) and CpG DNA motifs ( <strong>chapter eight</strong> ). Carp infected with <em>T. borreli</em> were found to upregulate the expression of the inflammatory cytokines TNF<FONT FACE="Symbol">a</font>and interleukin (IL)-1<FONT FACE="Symbol">b</font>early during infection. During a later phase, an upregulation of acute phase proteins (serum amyloid A, complement factor 3 and alpha-2-macroglobulin) was seen. Infection with <em>T. borreli</em> induced a non-specific proliferation of lymphocytes, most probably via the induction of TNF<FONT FACE="Symbol">a</font>and IL-1<FONT FACE="Symbol">b</font>, leading to the formation of parasite-aspecific antibodies. However, late during infection trypanotolerant carp do produce specific antibodies that act together with complement in lysing <em>T. borreli</em> .Stress, imposed by daily handling, severely affected resistance of carp to <em>T. borreli</em> ( <strong>chapter 9</strong> ). Most likely, the effect was mediated by increased levels of cortisol. We demonstrated that, <em>in vitro,</em> cortisol inhibited <em>T. borreli</em> -induced expression of IL-1<FONT FACE="Symbol">b</font>, TNF<FONT FACE="Symbol">a</font>, SAA and iNOS thereby modulating the immune response. Cortisol also induced apoptosis of lymphocytes, but not of phagocytes. One of the first cellular metabolic changes during cortisol-induced apoptosis was a depletion of GSH. As GSH plays a major role in the protection against NO-mediated inhibition of lymphocyte proliferation, cortisol may render stressed animals more susceptible to the immunopathological effects of NO.In conclusion, infection of carp with blood flagellates presents an excellent model for comparative studies on host-parasite interactions. Evaluation of the modulating effects of stress on the immune response to this type of pathogens can provide information with clear relevance to the disease problems faced by intensive animal production systems.