Pilar Alvarez-Pellitero

Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects

The increasing economic importance of fish parasitoses for aquaculture and fisheries has enhanced the interest in the defence mechanisms against these infections. Both innate and adaptive immune responses are mounted by fish to control parasite infections, and several mechanisms described for mammalian parasitoses have also been demonstrated in teleosts. Innate immune initiation relies on the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognizing receptors (PRRs). A number of PRRs, mainly Toll-like receptors (TLRs), have been characterized in fish, and some molecules susceptible of functioning as PAMPs are known for some fish parasites. A lectin-carbohydrate interaction has also been described in some host fish-parasite systems, thus probably involving C-type lectin receptors. Inflammatory reactions involving cellular reactions, as phagocytosis and phagocyte activity (including oxidative mechanisms), as well as complement activity, are modulated by many fish parasites, including mainly ciliates, flagellates and myxozoans. Besides complement, a number of humoral immune factors (peroxidases, lysozyme, acute-phase proteins) are also implicated in the response to some parasites. Among adaptive responses, most data deal with the presence of B lymphocytes and the production of specific antibodies (Abs). Although an increasing number of T-cell markers have been described for teleosts, the specific characterization of those involved in their response is far from being obtained. Gene expression studies have demonstrated the involvement of other mediators of the innate and adaptive responses, i.e., cytokines [interleukins (IL-1, IL-8), tumor necrosis factor (TNF), interferon (IFN)], chemokines (CXC, CC), as well as several oxidative enzymes [inducible nitric oxide synthase (iNOS), cyclo-oxygenase 2 (COX-2)]. Information is scarcer for factors more directly linked to adaptive responses, such as major histocompatibility (MH) receptors, T cell receptors (TCRs) and IgM. Expression of some immune genes varied according to the phase of infection, and proinflammatory cytokines were mainly activated in the early stages. Gene expression was generally higher in the target tissues for some skin and gill parasites, as Ichthyophthirius multifiliis, Neoparamoeba spp. and Lepeophtheirus salmonis, thus confirming the relevance of mucosal immunity in these infections. The existence of protective responses has been demonstrated for several fish parasites, both in natural infections and in immunization studies. Most information on the mechanisms involved in protection deals with the production of specific Abs. Nevertheless, their levels are not always correlated to protection, and the precise involvement of immune mechanisms in the response is unknown in many cases. No commercial vaccine is currently available for piscine parasitoses, although experimental vaccines have been assayed against I. multifiliis, Cryptobia salmositica and scuticociliates. The known information points to the need for integrated studies of the mechanisms involved in protection, in order to choose the optimum antigen candidates, adjuvants and formulations.

Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species, Sparus aurata L. and Dicentrarchus labrax L.

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.

Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data

A new Myxozoa species causing enteritis and death in cultured turbot, Scophthalmus maximus, is described at light and electron microscope levels. In addition, small subunit ribosomal RNA gene sequences (SSU rDNA) from the new species and from similar myxozoans were obtained and used for phylogenetic inference, as complementary criteria to resolve its taxonomic classification. The new parasite is closely related to Myxidium leei, another enteric histozoic species from marine fish. However, the ascription of M. leei to the genus Myxidium was based on weak morphological evidence and is not supported by our rDNA data analysis. A close relationship with Zschokkella, the other morphologically related myxozoan genus is also not supported. The combined morphological and molecular study results in the establishment of the new genus Enteromyxum to accommodate the new species E. scophthalmi, and the former M. leei, which is transferred to the new genus as Enteromyxum leei (Diamant, Lom & Dyková 1994) n. comb. This genus of marine, histozoic and enteric myxozoans includes significant parasite species for marine finfish culture.

Chronic exposure to the parasite Enteromyxum leei (Myxozoa: Myxosporea) modulates the immune response and the expression of growth, redox and immune relevant genes in gilthead sea bream, Sparus aurata L.

The myxosporean parasite Enteromyxum leei invades the intestine of gilthead sea bream producing a slow-progressing disease, which may end in the death of fish. The present work aimed to better know the host immune response and the underlying molecular mechanisms, which may help to understand why some individuals seem to be refractory to the disease. Three main aspects involved in fish health and welfare (immune, growth and redox status) were studied in fish exposed to E. leei-contaminated effluent, in comparison with control animals (not exposed to the disease). After chronic exposure (113days), prevalence of infection was 67.8%. Among exposed fish, parasitized and non-parasitized fish exhibited clear differences in some of the measured innate immune factors (respiratory burst, serum peroxidases, lysozyme and complement), and in the expression of immune, antioxidant and GH-related genes. The respiratory burst of parasitized fish was significantly higher, and serum peroxidases and lysozyme were significantly decreased both in parasitized and non-parasitized fish. The gene expression of GHR-I, GHR-II, IGF-I and IGF-II was measured in head kidney (HK) samples, and that of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, alpha-2M, GR, GPx-1 and GRP-75 was measured in intestine and HK samples, by rtqPCR. Parasitized fish exhibited a down-regulation of IL-1beta, TNF-alpha and GPx-1 in the intestine, and GHR-I and IGF-I were also down regulated in HK. alpha-2M and GRP-75 were over-expressed in the intestine of parasitized animals. Non-parasitized fish had increased transcripts of GHR-I and IGF-I with respect to control animals, which could furnish their immunocytes with an advantage to combat the parasite. The expression of GHR-II and IGF-II was not altered by the parasite challenge.

Experimental transmission of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of turbot Scophthalmus maximus.

Several experiments were designed to elucidate the modes of transmission of the myxozoan parasite Enteromyxum scophthalmi to turbot Scophthalmus maximus. Direct transmission of the infections was achieved by cohabitation of infected and test fish, through waterborne contamination from the effluent of a tank containing infected fish, and via the oral route using parasite-infected intestines. The transmission of the turbot enteromyxosis was successful in all the fish exposed to the parasite by the 3 routes; accumulated mortality reached 100% at the end of most experiments. The progress of the infections was monitored by study of the histopathology. Influence of the mode of exposure was observed, with the oral route the fastest to initiate the parasite infections. The temperature also affected the course of the infections, which were established earlier at higher water temperature. Direct fish-to-fish transmission of the disease explains the rapid spreading of the turbot enteromyxosis in farms.

Growth hormone as an in vitro phagocyte-activating factor in the gilthead sea bream (Sparus aurata)

Abstract.The stimulatory action of growth hormone on gilthead sea bream phagocyte-enriched cultures was demonstrated in vitro for the first time in a fish species. Phagocytes consisted mainly of macrophages, with a small number of neutrophils and eosinophils. Macrophages were unequivocally identified by their esterase staining and the lack of myeloperoxidase staining. Cells primed with recombinant rainbow trout GH showed clear morphological (light- and scanning electron-microscopic) and functional differences from non-primed cells. Stimulated phagocytes exhibited numerous branched lamellipodia, abundant membrane ruffles, increased spreading, and cell size. When incubated with sheep red blood cells, the phagocytic index and phagocytic capacity was also enhanced in primed cells. A bell-shaped dose-response curve (1.5-500 nM) was obtained when the metabolic activity of growth-hormone-activated cells was measured. This finding suggests that the homodimerization of the growth hormone receptor is a characteristic feature both in mammals and fish.

Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei).

Enteromyxum leei is an intestinal parasite responsible for serious outbreaks in Mediterranean sharpsnout sea bream Diplodus puntazzo. E. leei infection was experimentally transmitted to healthy D. puntazzo (R) by cohabitation with infected donor fish. Haematological changes and histopathological damage were evaluated in relation to the course of infection. The prevalence of infection in R fish was 100% from day 10 post-exposure (p.e.) onwards, and the infection intensity and histopathological damage increased progressively. Different developmental stages were found in the infected intestines, including proliferative (stages 1-3) and sporogonic (stages 4 and 5) stages. Intestinal damage consisted of vacuolation, necrosis, detachment and sloughing of epithelium, and was correlated with the progression of the infection and with the development of the parasite. Sporogonic stages appeared from day 20 p.e. onwards. Initially, D. puntazzo seems to counteract the infection through the increase in leucocyte numbers, respiratory burst activity, haematopoietic activity and MMC. Two types of eosinophilic granular cells (EGC1 and EGC2) were detected in the intestinal epithelium and lamina propria. EGC1 numbers decreased with the progression of infection, whereas an increase in EGC2 occurred, mainly in the lamina propria. The involvement of the cellular immunity in the response of D. puntazzo to E. leei was demonstrated. The depletion of this response at a certain point of the infection could contribute to the high virulence of this myxozoan in this fish species.

Gilthead seabream ( Sparus aurata L.) innate defence against the parasite Enteromyxum leei (Myxozoa)

The humoral innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei has been studied. At 10, 22, 38, 52 and 108 days of cohabitation fish were sampled to examine gut histology and to determine serum innate immune parameters and the mRNA expression of pro-inflammatory cytokines (IL-1beta and TNFalpha) in head-kidney. The parasite was successfully transmitted to 45% of the recipient fish and prevalence reached a maximum (62.5%) at the last sampling time (108 days). Recipient fish started to die after 74 days of cohabitation. In general, alternative complement activity was higher whereas the peroxidase level was lower in recipient fish than in controls. Moreover, IL-1beta mRNA expression increased while the TNFalpha gene expression decreased in recipient fish. These data demonstrate the involvement of complement activity in the defence mechanisms of the gilthead seabream against the myxosporean E. leei. Within the recipient fish group, few differences were observed in the studied immune parameters between E. leei-parasitized and non-parasitized recipient fish. Parasitological and immunological implications of E. leei infections in Mediterranean fish farms are discussed.

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass.

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.

Cellular and humoral immune response of European sea bass ( Dicentrarchus labrax L.) (Teleostei: Serranidae) immunized with Sphaerospora dicentrarchi (Myxosporea: Bivalvulida)

The immune response of European sea bass after intracaelomic immunization with Sphaerospora dicentrarchi was studied. Fish were injected with S. dicentrarchi spores (DIC), with spores plus adjuvant (DIC + FCA), with adjuvant alone (FCA) or with PBS. Several parameters of the immune response were measured. Serum lysozyme increased significantly in DIC fish 1 week after immunization (p.i.) and it remained significantly higher in DIC + FCA fish 4 weeks p.i., and in DIC fish 8 weeks p.i. than in PBS-injected fish. The number of nitroblue tetrazolium-positive blood cells was significantly higher in DIC + FCA fish 1, 4 and 8 weeks p.i, but the highest values were detected 1 week p.i. The highest stimulation index was detected in phagocytes from DIC + FCA fish. The number of S. dicentrarchi antibody-secreting cells was significantly higher in DIC + FCA fish than in DIC fish. Serum from DIC and DIC + FCA fish, stained the polar capsules and the valves of S. dicentrarchi spores in immunohistochemistry. Serum antibodies could not be detected using immunoblot assay. All these results show that immunization with S. dicentrarchi resulted in the activation of the non-specific immune response, mainly 7 days p.i. A specific humoral response against the parasite was also demonstrated but it had a low magnitude.