For many years hexamitids, Hexamita spp. and Spironucleus spp., have frequently been reported in vertebrates, particularly in fish. This suggests a potentially important role of these parasites in the fish culture industry. Though the majority of hexamitids are not known to cause disease in their vertebrate host, those that have been documented as associated with disease are still in need of further investigation into their geographical distribution, host range, life cycle, host-parasite relationship, pathogenicity, diagnosis, prevention, treatment, and control. Spironucleus vortens is a hexamitid recently described from angelfish (Pterophyllum scalare). Although the structure of this parasite has been investigated using the electron microscope (Poynton et al., 1995), other information on this organism is poorly understood. Thus, the purpose of this research was to study the nature of S. vortens in TYI-S-33 culture medium and in the angelfish host. The optimal environmental conditions for S. vortens growth were investigated using variations of temperature, pH, and bile concentrations. This study is useful in helping to understand the locations and environmental conditions in the host that are suitable for the growth of S. vortens. Treatment of S. vortens, using seven chemotherapeutic agents; dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole, and magnesium sulfate was evaluated. The pathogenicity of S. vortens in angelfish was investigated in fish experimentally inoculated with trophozoites. This study provided information to help understand the pathogenesis of the parasites in the host. Finally, to examine the protective defense mechanisms, the presence of anti-S. vortens antibodies in angelfish serum were evaluated along with the presence of immune cells (lymphocytes, macrophages, eosinophilic granular cells, neutrophils, and plasma cells) at invaded sites of the intestine and other internal organs in response to an experimental Spironucleus vortens infection. The results of this research provide information on this parasite's effect on the fish host which may be useful in understanding the nature of other hexamitids. A few published reports have suggested the in vitro growth requirement of fish Spironucleus (Poynton et al., 1995; Sterud, 1998), but none have examined the optimal conditions required for growth and the pathogenicity of S. vortens. The first study was to examine the optimal requirements for the in vitro growth of the parasite. The organisms were cultivated in either an artificial medium (TYI-S-33) at different temperatures or various pH conditions, or in medium supplemented with different bile concentrations at 25°C. Criteria used to justify the optimal conditions were average cell number ml-1, growth rate, survival time, and cell conditions (motility and morphology). The organisms survived longest at 22°C, and had the highest average cell number ml-1 at 25°, 28° and 31°C. At 25°C the parasites were highly active and survived up to 6 days. The organisms cultivated at pH 6.5, 7.0 and 7.5 yielded the highest average cell number ml-1 with survival periods up to 13-14 days. Most of the organisms cultivated at a pH lower than 6.0 or a pH higher than 7.5 were suppressed and killed within 5-6 days of cultivation. All cultures supplemented with bovine or fish bile yielded lower maximal numbers of parasites than cultures with no bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25°C and pH 6.5 to 7.5 without supplementation with bile. In order to treat spironucleosis, the efficacy of various chemotherapeutic agents on the growth of S. vortens was examined in vitro. In this study nitroimidazoles and benzimidazoles, formerly reported as drugs of choice for the treatment of diplomonads, pyrimethamine and magnesium sulfate (Epsom salt) were evaluated at different concentrations on the growth of S. vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth at concentrations of 1 μg ml-1 or higher. Albendazole and fenbendazole suppressed the growth of parasites at concentrations of 1.0 μg ml-1 or higher after 24 h exposure. Mebendazole was the most effective agent of the benzimidazole group; and inhibited the parasite's growth at concentrations of 0.5 μg ml-1 or higher. Pyrimethamine at concentrations of 1-10 μg ml-1 failed to inhibit the parasite's growth. Magnesium sulfate inhibited the growth of the parasites only at high concentrations (70 mg ml-1 or higher) . This study indicates that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens. To investigate the pathogenesis of spironucleosis, angelfish were orally (PO) or intraperitoneally (IP) inoculated with S. vortens. Control angelfish which were orally gavaged or intraperitoneally injected with PBS were in normal body condition and had no morbidity or mortality. Compared to the control angelfish, PO-infected angelfish were inappetent with no other clinical signs, while IP-infected angelfish showed clinical signs of inappetite, weakness, respiratory distress, and laying on their sides. Twenty percent of the IP-infected angelfish died within the first three weeks after infection. In PO-infected angelfish, the organisms were located only in the intestinal lumen. In IP-infected angelfish, S. vortens were found in the blood, stomach, intestine, and other internal organs (spleen, gall bladder, and ovary). However, no parasites were observed within the intestinal mucosa of either PO- or IP-infected fish. Histopathologic examination of the intestines revealed mild to moderate multifocal enteritis in both PO- and IP-infected angelfish. The mucosal epithelium appeared undamaged although the parasite was closely located and appeared attached to the intestinal mucosa. The results suggest that S. vortens normally causes mild to moderate multifocal enteritis with no morbidity. However, the parasites can cause granulomatous inflammation in a wide variety of host tissues, and may be lethal if they enter the abdominal cavity and disseminate to other organs via the blood circulation. Immunity, both cell mediated and humoral, against S. vortens was investigated in this study. Histopathologic examination revealed a response from inflammatory cells infiltrated and localized in the affected tissues. Macrophages, lymphocytes, and plasma cells were the most common cell types found in the internal organs. Macrophages were active in the affected tissues where the parasites lived in situ. However, in vitro studies indicated that there were no differences in a production of H₂O₂ or in phagocytosis between macrophages of control and infected angelfish regardless of inoculum dosage and administration route. A preliminary study of humoral antibody indicated that angelfish did not develop anti-S. vortens antibody after they were orally or intraperitoneally infected with either a low or a high number of the organisms. It is suggested that localized leucocyte response may be an important mechanism against Spironucleus vortens infection in angelfish. This research has indicated some of the important environmental factors affecting the parasite's growth, and has provided some initial information on the pathogenicity of S. vortens. In addition, preliminary information on the host's protective immune systems, humoral and cell-mediated immunity, against the parasite have been documented. The results from this research will be useful for aquaculture, particularly of tropical freshwater angelfish, and may help to provide an understanding of the biological roles of other hexamitids. / Ph. D.

Made available in DSpace on 2014-07-29T15:13:51Z (GMT). No. of bitstreams: 1 Tese_Wallace_Oliveira.pdf: 492446 bytes, checksum: fe7d8543a910f45c0ceb93e5a9c93bb8 (MD5) Previous issue date: 2009-05-29 / In order to increase the knowledge on the production of angelfish ornamental cichlids (Pterophyllum scalare), two experiments were conducted at the School of Veterinary Medicine and Zootecnia of the UFT, Araguaína /TO, between october 2008 and january 2009, using 400 angelfish juveniles. Each experiment had a duration of 42 days, with fish divided into 16 aquariums, fed ad libitum, room temperature, 20% daily water renewal. The work was organized in a factorial scheme 2x2, with: two levels of crude protein (CP, 29 and 32%) and two angelfish strains (gold and marmorate), with 160 juveniles from 350+4.0 mg in design randomized blocks, in the first experiment; two levels of digestible energy (DE, 2.9 and 3.4 Mcal / kg) and two stocking rate (10 or 20 fish/aquarium), with 240 fishes of 260+4.2 mg in a completely randomized design in the second. Means were compared by Tukey test at 5%. Weight gain (WG), apparent feed intake (AFI), food conversion rate (FCR), protein efficiency rate (PER), specific growth rate in weight (WSGR) and length (LSGR), Fulton condition factor and carcass composition were avaliated. It was found that the levels of CP did not differ in the parameters studied (P>0.05) and the marmorate strain was more efficient than gold strain by presenting best means of WG, AFI, FCR, PER, WSGR, LSGR (P<0.05). Also, fish fed 3.4 Mcal of DE/kg showed better means WG, AFI, FCR, PER, WSGR and LSGR (p<0.05). There was no significant difference in parameters of growth, consumption and conversion between the stocking rate (p>0.05). It was concluded that angelfish juveniles have similar performance when fed with 29 or 32% CP and better in the diets with 3.4 Mcal of DE/kg than with 2.9 Mcal/kg. Also, marmorate strain was better than gold strain and at this stage of development, can be created in stocking rate of one fish for every three liters of water, with 20% of daily water renewal / Com o objetivo de se ampliar o conhecimento sobre a produção do ciclídeo ornamental acará-bandeira (Pterophyllum scalare), dois experimentos foram realizados na Escola de Medicina Veterinária e Zootecnia da UFT, Araguaína/TO, entre outubro de 2008 e janeiro de 2009, utilizando-se 400 juvenis de acará-bandeira. Cada experimento teve a duração de 42 dias, com peixes divididos em 16 aquários, alimentados à vontade, temperatura ambiente, renovando-se 20% da água/dia. Os trabalhos foram organizados em esquema fatorial 2x2, sendo: dois níveis de proteína bruta (PB; 29 e 32%) e duas linhagens de acará-bandeira (ouro e marmorato), com 160 juvenis de 350+4,0 mg, em delineamento de blocos casualizados no primeiro experimento; dois níveis de energia digestível (ED; 2,9 e 3,4 Mcal/kg) e duas densidades de estocagem (10 ou 20 peixes/aquário), com 240 peixes de 260+4,2 mg, em delineamento inteiramente casualizado, no segundo experimento. Em ambos, as médias foram comparadas pelo teste de Tukey, a 5%. Avaliou-se ganho de peso (GP), consumo aparente de ração (CAR), conversão alimentar aparente (CAA), taxa de eficiência protéica (TEP), taxa de crescimento específico em peso (TCEP) e em comprimento (TCEC), fator de condição de Fulton e composição da carcaça. Verificou-se que os níveis de PB não diferiram nos parâmetros estudados (P>0,05) e que a linhagem marmorato foi mais eficiente do que a linhagem ouro por apresentar melhores médias de GP, CAR, CAA, TEP, TCEP, TCEC (P<0,05). Também, que os peixes alimentados com 3,4 Mcal de ED/kg apresentaram melhores médias de GP, CAR, CAA, TEP, TCEP e TCEC (p<0,05). Não houve diferença significativa nos parâmetros de crescimento, consumo e conversão de alimento entre as densidades de estocagem (p>0,05). Concluiu-se que juvenis de acará-bandeira apresentam desempenho semelhante quando alimentados com 29 ou 32% de PB e melhor em dietas com 3,4 Mcal de ED/kg do que com 2,9 Mcal/kg. Também, que os da linhagem marmorato desenvolvem-se melhor, comparados aos da linhagem ouro e que, nesta fase de desenvolvimento, podem ser criados na densidade de um peixe para cada três litros de água, com 20% de renovação diária da água