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Diplostomiasis, or eyefluke is caused by the metacercarial stage of the digenean parasite <i>Diplostomum spathaceum</i> which selectively invades the lenses of freshwater fish and can cause partial and total blindness. This study investigates the host-parasite relationship of <i>D. spathaceum</i> infections in rainbow trout with particular reference to the diplostomule or post-penetration stage of the parasite. The point of cercarial penetration influenced the speed with which diplostomules reached the lens and the length of time they were exposed to the fish defence mechanisms. The further away the point of cercarial penetration from the eye, the longer diplostomules take to reach the lens and the fewer establish as metacercariae. Total body immersion using cercariae resulted in significantly more metacercariae establishing in the lens compared with entry via intramuscular, intraperitoneal or intracardial injection of either cercariae or diplostomules. Cercariae may be drawn in by the respiratory current and enter the fish via the gills and buccal cavity. The gills provide an immediate source of entry into the circulatory and lymphatic system of the fish. There did not appear to be any preferred route of migration. The optimum conditions for the <i>in vitro</i> collection of diplostomules were developed. The optimum conditions for <i>in vitro</i> transformation of <i>D. spathaceum</i> cercariae were established. The optimum conditions for the short-term <i>in vitro</i> maintenance of diplostomules were investigated. L-15 medium supplemented with 5% heat-inactivated foetal calf serum maintained approximately 80% survival for the first 24-48 h of culture. Rainbow trout were immunized with cercariae and diplostomules of <i>D. spathaceum</i>. Circulating antibody was detected by ELISA to both cercariae and diplostomules by 6-7 weeks post-immunization. Cross-reactivity occurred between anti-cercarial serum and diplostomule and metacercarial antigens, and anti-diplostomule serum cross-reacted with cercarial and metacercarial antigens in ELISA. Immunofluoresence studies indicated that the tail region of the cercriae was strongly antigenic; a differential fluorescence was observed for both anti-cercarial and anti-diplostomule sera with the cercarial tails fluorescing more strongly than the bodies. No cross-reactivity was observed in fluorescence between anti-cercarial or anti-diplostomule sera and metacercarial antigen although anti-cercariae serum cross-reacted with diplostomule antigen and <i>vice versa</i>. Normal trout serum and anti-diplostomule serum were cytotoxic to diplostomules of <i>D. spathaceum in vitro</i>. Immunized fish exhibited a significant degree of protection against further infection by <i>D. spathaceum</i>. However, there was no correlation between protection and the level of serum antibody. Isolated normal trout macrophages kill significant numbers of diplostomules <i>in vitro</i> at effector: parasite ratios of 150:1 or greater. <i>In vitro</i> killing was not increased using diplostomules opsonized with anti-serum or <i>in vivo</i>-activated macrophages individually, but when combined, increased killing occurred. Diplostomules were capable of eliciting respiratory burst activity from macrophages <i>in vitro</i>, suggesting that reactive oxygen species may have a role to play in the killing mechanism. Diplostomule antigen was capable of activating macrophages <i>in vivo</i>.

The ability of thick-lipped grey mullet to mount both humoral and cellular immune responses to Cryptocotyle lingua has been demonstrated here for the first time. Of the 3 procedures for immunisation investigated, namely exposure to live cercariae, intraperitoneal (ip) injection of whole killed cercariae and ip injection of a cercarial sonicate, the former gave the highest primary antibody titres with a peak response at 4 weeks post infection. Peak titres occurred at 5 and 7 weeks after immunisation by ip injection of sonicated cercariae and whole killed cercariae respectively. An enhanced secondary antibody response was observed following challenge by ip injection of sonicated cercariae. Cercariacidal activity, apparently involving complement, was detected in normal serum and enhanced in immune serum. However, the mechanism for complement activation remains uncertain. Parasite antigens were identified following separation by SOS-PAGE and immunoblotting using mullet and rabbit antisera. Mullet failed to respond to certain parasite proteins recognised by rabbit antiserum and in situ immunogold-silver staining demonstrated that mullet. immunised by exposure to live cercariae, were unable to recognise the metacercarial cyst wall. Migration and polarisation of pronephric leucocytes in vitro was enhanced following immunisation but cellular adherence to encysted and unencysted parasites was not observed. In vitro studies further demonstrated a possible role for fish serum in stimulating metacercarial encystment. The intensity of melanisation of the host capsule in metacercarial infections was increased in fish held in total darkness and with increased temperature. The rate of development of the metacercarial tegument and cyst wall and the associated host response was similarly affected by temperature and initial development was inhibited by prior immunisation via ip injection of sonicated cercariae. There was however, no evidence of protection against cercarial infection following such immunisation. These results are discussed in relation to mechanisms of immunity, metacercarial survival strategies and control of metazoan infections in fish.

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Orientador: Reinaldo José da Silva / Coorientador: Sérgio Henrique Canello Schalch / Banca: Eduardo Makoto Onaka / Banca: Maurício Laterça Martins / Resumo: O desenvolvimento das atividades relacionadas à aquicultura tem aumentado consideravelmente a relevância dos estudos sobre parasitas e outros patógenos de organismos aquáticos, principalmente, daqueles hospedeiros com potencial para o cultivo e comercialização. O objetivo deste estudo foi avaliar a ocorrência de infecções parasitárias e microbianas na produção de pacu Piaractus mesopotamicus e do híbrido "patinga" (P. mesopotamicus x Piaractus brachypomus) procedentes da região Noroeste do Estado de São Paulo, Brasil. Três pisciculturas foram amostradas bimestralmente, de fevereiro de 2010 a fevereiro de 2011: A - Reprodução e Larvicultura (pacu/patinga), B - Engorda (patinga) e C - Pesque-pague (pacu/patinga), das quais 35 peixes foram submetidos à análise parasitológica e 21 à análise microbiológica. Foram encontrados os parasitas: dactilogirídeos (Monogenea); Trichodina spp., Ichthyophthirius multifiliis, Chilodonella sp. (Protozoa); Myxobolus spp. e Henneguya spp. (Myxozoa); Rondonia rondoni e Contracaecum sp. (Nematoda); e Dolops carvalhoi (Crustacea). Dentre os peixes analisados, 62,9% de "A" e 100% de "B" e "C" estavam infectados/infestados por pelo menos uma espécie de parasita. Pacus (n=44) apresentaram maior suscetibilidade a infestações por Anacanthorus penilabiatus, e as patingas (n=61), por Mymarothecium viatorum (p<0,05). Na análise microbiológica, 23,8% dos peixes de "A" e "B" e 33,3% da propriedade "C" estavam infectados com pelo menos um gênero de bactéria. A sazonalidade influenciou as infestações por Trichodina spp., A. penilabiatus e infecções pela bactéria Enterobacter sp. em patingas (p<0,05). No antibiograma, cepas de Pseudomonas sp., Stenotrophomonas maltophilia e Aeromonas sp. apresentaram resistência a todos antibióticos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The development of activities related to aquaculture has considerably increased the relevance of studies on parasites and other pathogens of aquatic organisms, especially those with potential for production and marketing. The aim of this study was to evaluate the occurrence of parasitic and microbial infections in the "pacu" Piaractus mesopotamicus and hybrid "patinga" (P. mesopotamicus x Piaractus brachypomus) from the Northwest of São Paulo State, Brazil. Fishes from three fish farms were evaluated every two months: A - Reproduction and larviculture (pacu / patinga), B - Growout (patinga) and C - Fee-fishing property (pacu / patinga). Fishes from each fish farm were collected from February 2010 to February 2011 for parasitological (n = 35 per property) and microbiological (n = 21 per property) analysis. The parasites found were: Dactylogyridae (Monogenea), Trichodina spp., Ichthyophthirius multifiliis, Chilodonella sp. (Protozoa), Myxobolus spp., Henneguya spp. (Myxozoa), Rondonia rondonia, third stage larvae of Contracaecum sp. (Nematoda), and Dolops carvalhoi (Crustacea). Among the fishes examined, 62.9% from "A" and 100% from "B" and "C" were infected / infested with at least one parasite species. Pacu (n = 44) showed higher susceptibility to infestations caused by Anacanthorus penilabiatus and patinga (n = 61) by Mymarothecium viatorum (p < 0.05). In the microbiological analysis, 23.8% of fishes from "A" and "B" properties and 33.3% of fish from the property "C" were infected with at least one bacteria species. Seasonality influenced Trichodina spp. and A. penilabiatus infestations and infection caused by Enterobacter sp. in patingas (p < 0.05). In antibiogram test, strains of Pseudomonas sp., Stenotrophomonas maltophilia and Aeromonas sp. were resistant to all beta-lactam antibiotics... (Complete abstract click electronic access below) / Mestre