O I. multifiliis é um protozoário ciliado que acomete diferentes espécies de peixes, causador da ictiofitiriase, responsável por altas taxas de mortalidade em pisciculturas e resultando em perdas econômicas. O presente estudo avaliou isolados do parasito oriundos de peixes capturados em seis estados brasileiros, utilizando como marcadores moleculares ribossomais os genes 18S, 5.8S e 28S e os espaçadores intergênicos ITS1 e ITS2 de I. multifiliis, obtidos através da Reação em Cadeia da Polimerase (PCR) e posteriormente, sequenciados. Primers específicos foram desenhados para este estudo. As sequências dos isolados de I. multifiliis encontrados infectando diferentes hospedeiros em diferentes estados brasileiros apresentaram grande similaridade e homologia, sendo muito conservadas e, por este fato, não permitem diferenciação entre isolados deste parasito. Sequências do DNA de isolados de I. multifiliis de diferentes regiões brasileiras foram depositadas no GenBank e representam as primeiras informações para esta espécie no Brasil. / The I. multifiliis is a ciliate protozoan that affects fishes of different species, which causes Ichthyophthiriasis. This disease is responsible for high mortality in fish farms resulting in economic losses. The present study evaluated parasite isolates from four Brazilian states, using nuclear ribosomal molecular makers for 18S,5.8S and 28S genes and the first and second internal transcribed ITS1 and ITS2 intergenic spacers from I. multifiliis. All the markers were evaluated through Polymerase Chain Reaction (PCR) and rDNA sequenced, using novel specific primers; the sequences of different isolated of I. multifiliis, which were collected from different fish species (host) from different Brazilian regions showed high similarity and homology, which were extremely conserved therefore it was not possible to differentiate among the strains from this parasite. All recovered sequences from this study were deposited in GenBank database. These sequences represent a novel contribution for Brazilian isolate of I. multifiliis.

O protozoário ciliado Ichthyophthirius multifiliis causador da doença dos pontos brancos em muitas espécies de peixes de água doce destaca-se como um importante patógeno por ser responsável por grandes perdas para a indústria de pescados mundial. Duas espécies de peixes de grande importância para a economia no Brasil foram selecionadas para este estudo, o pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) e a cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). Com o objetivo de avaliar a resposta imune destes peixes frente ao parasita, inicialmente, o protozoário I. multifiliis foi isolado e repicado em P. corruscans para a obtenção da forma infectante (teronte) utilizada nas imunizações. A identificação molecular dos peixes foi realizada através da amplificação e sequenciamento do gene RAG2 mostrando que as espécies puras são homozigotas enquanto o híbrido é heterozigoto para este gene. Através de PCR foi realizada ainda a caracterização parcial dos genes arginase 1, arginase 2, IL-1β, IL-8 e TNF-α dos peixes pertencentes a família Pseudoplatystoma que resultaram em amplificações de sequências com aproximadamente 530, 815, 400, 280 e 300 pb, respectivamente, que foram usadas para a construção de primers específicos utilizados nas análises de expressão gênica por PCR em tempo real (qPCR). A expressão gênica em P. corruscans e P. reticulatum imunizados com terontes vivos de I. multifiliis foram verificadas 6, 12, 18 e 24 horas após imunização, sendo retirada amostras do baço, fígado e rim anterior dos peixes. No geral a expressão dos genes destes órgãos em P. corruscans apresentam um padrão semelhante de regulação. Um aumento significativo de expressão em relação ao período de imunização foi verificado para: a arginase 2 as 6 e 18 horas no baço, as 6 h no fígado e as 6, 18 e 24 horas no rim cranial; o gene da IL1-β as 24 h no fígado e 18 h e 24 h no rim cranial; e o gene da IL-8 somente as 18 h no baço. Já os genes arginase 1 e TNFα não tiveram regulação significativa nos períodos. Para P. reticulatum observou-se que somente o baço teve aumento na regulação com diferenças significativas nos períodos para arginase 2, IL-8 e TNFα, as 6 e 18 horas, já os genes arginase 1 e IL1-β não apresentaram alterações significativa. / The ciliate protozoan Ichthyophthirius multifiliis has been reported in various freshwater fishes and stands out as an important pathogen for being responsible to severe losses to both food and aquarium fish production. Among the fish species explored economically in Brazil it can be highlight the pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) and cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). At this study the parasite I. multifiliis was original isolated from a petshop infected fish and maintained by serial transmission on naive pintado (P. corruscans) to obtain the infecting form (teronte) used at immunizations. The molecular identification of fish was realized through the amplification and sequencing of RAG2 gene, were the pintado (P. corruscans) and cachara (P. reticulatum) were homozygous, while the hybrid between those fish was considered heterozygous for this gene. At the partial gene characterization from fishes belonging to the Pseudoplatystoma family, the PCR amplification result in sequences of 530, 815, 400, 280 and 300 pb of the genes arginase 1, arginase 2, IL1-β, IL-8 e TNFα respectively, which were used to construct of specific primers for the quantitative real-time PCR (qPCR). The gene expression at P. corruscans and P. reticulatum immunized with live theronts of I. multifiliis was evaluated 6, 12, 18 and 24 hours after the immunization, where it was collected samples of the spleen, liver and heady kideny. The gene expression of the organs of pintado (P. corruscans) showed similar pattern of expression. An up-regulation with statistical significance was observed for: arginase 2 at 6 and 18 hours at spleen, 6 h at kidney and 6, 18 and 24 h at head kidney; the gene of IL1-β at 24 h at spleen, 18 and 24 hours at head kidney; the gene of IL-8 at 18 h at spleen. The genes of arginase 1 and TNFα didn\'t showed statistical significant regulation at this experiment. At the gene expression of cachara (P. reticulatum) only the spleen had a statistical alteration after theronts immunization where the arginase 2, IL-8 and TNFα was up-regulated at 6 h and 18 h, while the arginase 1 and IL1-β didn\'t showed statistical alteration.

O I. multifiliis é um protozoário ciliado que acomete diferentes espécies de peixes, causador da ictiofitiriase, responsável por altas taxas de mortalidade em pisciculturas e resultando em perdas econômicas. O presente estudo avaliou isolados do parasito oriundos de peixes capturados em seis estados brasileiros, utilizando como marcadores moleculares ribossomais os genes 18S, 5.8S e 28S e os espaçadores intergênicos ITS1 e ITS2 de I. multifiliis, obtidos através da Reação em Cadeia da Polimerase (PCR) e posteriormente, sequenciados. Primers específicos foram desenhados para este estudo. As sequências dos isolados de I. multifiliis encontrados infectando diferentes hospedeiros em diferentes estados brasileiros apresentaram grande similaridade e homologia, sendo muito conservadas e, por este fato, não permitem diferenciação entre isolados deste parasito. Sequências do DNA de isolados de I. multifiliis de diferentes regiões brasileiras foram depositadas no GenBank e representam as primeiras informações para esta espécie no Brasil. / The I. multifiliis is a ciliate protozoan that affects fishes of different species, which causes Ichthyophthiriasis. This disease is responsible for high mortality in fish farms resulting in economic losses. The present study evaluated parasite isolates from four Brazilian states, using nuclear ribosomal molecular makers for 18S,5.8S and 28S genes and the first and second internal transcribed ITS1 and ITS2 intergenic spacers from I. multifiliis. All the markers were evaluated through Polymerase Chain Reaction (PCR) and rDNA sequenced, using novel specific primers; the sequences of different isolated of I. multifiliis, which were collected from different fish species (host) from different Brazilian regions showed high similarity and homology, which were extremely conserved therefore it was not possible to differentiate among the strains from this parasite. All recovered sequences from this study were deposited in GenBank database. These sequences represent a novel contribution for Brazilian isolate of I. multifiliis.

O protozoário ciliado Ichthyophthirius multifiliis causador da doença dos pontos brancos em muitas espécies de peixes de água doce destaca-se como um importante patógeno por ser responsável por grandes perdas para a indústria de pescados mundial. Duas espécies de peixes de grande importância para a economia no Brasil foram selecionadas para este estudo, o pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) e a cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). Com o objetivo de avaliar a resposta imune destes peixes frente ao parasita, inicialmente, o protozoário I. multifiliis foi isolado e repicado em P. corruscans para a obtenção da forma infectante (teronte) utilizada nas imunizações. A identificação molecular dos peixes foi realizada através da amplificação e sequenciamento do gene RAG2 mostrando que as espécies puras são homozigotas enquanto o híbrido é heterozigoto para este gene. Através de PCR foi realizada ainda a caracterização parcial dos genes arginase 1, arginase 2, IL-1β, IL-8 e TNF-α dos peixes pertencentes a família Pseudoplatystoma que resultaram em amplificações de sequências com aproximadamente 530, 815, 400, 280 e 300 pb, respectivamente, que foram usadas para a construção de primers específicos utilizados nas análises de expressão gênica por PCR em tempo real (qPCR). A expressão gênica em P. corruscans e P. reticulatum imunizados com terontes vivos de I. multifiliis foram verificadas 6, 12, 18 e 24 horas após imunização, sendo retirada amostras do baço, fígado e rim anterior dos peixes. No geral a expressão dos genes destes órgãos em P. corruscans apresentam um padrão semelhante de regulação. Um aumento significativo de expressão em relação ao período de imunização foi verificado para: a arginase 2 as 6 e 18 horas no baço, as 6 h no fígado e as 6, 18 e 24 horas no rim cranial; o gene da IL1-β as 24 h no fígado e 18 h e 24 h no rim cranial; e o gene da IL-8 somente as 18 h no baço. Já os genes arginase 1 e TNFα não tiveram regulação significativa nos períodos. Para P. reticulatum observou-se que somente o baço teve aumento na regulação com diferenças significativas nos períodos para arginase 2, IL-8 e TNFα, as 6 e 18 horas, já os genes arginase 1 e IL1-β não apresentaram alterações significativa. / The ciliate protozoan Ichthyophthirius multifiliis has been reported in various freshwater fishes and stands out as an important pathogen for being responsible to severe losses to both food and aquarium fish production. Among the fish species explored economically in Brazil it can be highlight the pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) and cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). At this study the parasite I. multifiliis was original isolated from a petshop infected fish and maintained by serial transmission on naive pintado (P. corruscans) to obtain the infecting form (teronte) used at immunizations. The molecular identification of fish was realized through the amplification and sequencing of RAG2 gene, were the pintado (P. corruscans) and cachara (P. reticulatum) were homozygous, while the hybrid between those fish was considered heterozygous for this gene. At the partial gene characterization from fishes belonging to the Pseudoplatystoma family, the PCR amplification result in sequences of 530, 815, 400, 280 and 300 pb of the genes arginase 1, arginase 2, IL1-β, IL-8 e TNFα respectively, which were used to construct of specific primers for the quantitative real-time PCR (qPCR). The gene expression at P. corruscans and P. reticulatum immunized with live theronts of I. multifiliis was evaluated 6, 12, 18 and 24 hours after the immunization, where it was collected samples of the spleen, liver and heady kideny. The gene expression of the organs of pintado (P. corruscans) showed similar pattern of expression. An up-regulation with statistical significance was observed for: arginase 2 at 6 and 18 hours at spleen, 6 h at kidney and 6, 18 and 24 h at head kidney; the gene of IL1-β at 24 h at spleen, 18 and 24 hours at head kidney; the gene of IL-8 at 18 h at spleen. The genes of arginase 1 and TNFα didn\'t showed statistical significant regulation at this experiment. At the gene expression of cachara (P. reticulatum) only the spleen had a statistical alteration after theronts immunization where the arginase 2, IL-8 and TNFα was up-regulated at 6 h and 18 h, while the arginase 1 and IL1-β didn\'t showed statistical alteration.

Call number: LD2668 .T4 1979 L48 / Master of Science

The intensification of freshwater aquaculture worldwide has facilitated the propagation of the parasitic ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876 commonly known as “fish white spot” or “Ich”. Ichthyophthirius multifiliis infections lead to high mortalities, generating significant economic losses in most cultured freshwater fish species worldwide. Until recently, malachite green was the chemical treatment traditionally used to control I. multifiliis infections. Its reclassification as carcinogenic to humans and its subsequent ban for use in food fish has left the industry without any suitable treatments. Currently, in-bath formaldehyde and sodium chloride treatments are the most common option used in farm systems to control I. multifliis infections. Given their low efficacy, however, they are not considered as sustainable long–term options. There is, therefore, an urgent necessity to find efficacious alternatives for controlling I. multifliis infections. The general aim of this research project was to improve the management of I. multifiliis infections in order to develop more comprehensive, environmentally friendly and sustainable therapeutic strategies for use in freshwater food fish aquaculture. The present PhD-thesis present first a literature review chapter providing an overview and critical assessment of chemotherapeutants and physical interventions tested within the last 30 years against I. multifiliis infections. The experimental worked consisted of a number of in vitro and in vivo trials were conducted using experimental scale flow-through, static tank systems and commercial scale raceways within a rainbow trout hatchery, in addition to molecular work on different isolates of the parasite. The results of this research are organised into three experimental chapters which describe the testing of chemical and non-chemical treatments against I. multifiliis infections and work undertaken to determine the most suitable molecular markers to identify I. multifiliis isolates. In the first experimental chapters, the possibility of efficiently controlling I. multifliis infections through the administration of novel environmentally-friendly chemical treatments (e.g. bronopol and peracetic acid-based products) was investigated. The results clearly showed that bronopol and peractic acid-based products have a strong biocidal/cytotoxic effect against all free-living stages of I. multifiliis (e.g. tomonts, cysts and theronts). The administration of high concentrations of bronopol (e.g. 20, 50 and 100 mg L-1) over short periods of exposure (e.g. 30 min) significantly reduced the survival of tomonts, cysts and theronts and delayed the development of I. multifiliis tomonts and cysts. Prolonged low concentrations of bronopol (e.g. 1 mg L-1) greatly reduced the survival of infective theronts, although such treatment did not affect the ability of surviving theronts to subsequently infect a host. When tested in vivo, the continuous prolonged exposure (e.g. 27 days) of low concentrations of bronopol (e.g. 2 and 5 mg L-1) had an impact on the population dynamics of I. multifiliis, this being demonstrated by a significant reduction in the number of trophonts developing within the fish. Low concentrations of bronopol (e.g. 2 mg L-1) administrated as a preventive treatment prior to infection also proved to be very successful at reducing the colonisation success of I. multifiliis. Peracetic acid administrated at low concentrations (e.g. 8, 12 and 15 mg L-1) over a short window of exposure (e.g. 1 h) displayed a strong biocidal effect against all the free-living stages of I. multifiliis (e.g. tomonts, cysts and theronts). The bronopol and peracetic acid-based products tested here both appear to be capable of disrupting the development of the cyst stage of I. multifiliis which is seldom reported for chemotherapeutants currently used against this parasite. These results suggest that bronopol and peracetic acid-based products have a place in the arsenal of treatment options for controlling I. multifiliis infections in commercial aquaculture systems. The use of a mechanical device or a biological control agent to remove the cyst stage of I. multifiliis and the impact of such control on the population dynamics and the levels of infection of fish were also investigated. The results revealed that tomonts preferentially settle and encyst on the base of culture systems and on biofilm–covered substrates. The survival of the tomont stage is greatly affected by the composition of the substrate upon which it settles and is significantly lower on polypropylene-based plastic. The lining of raceways in a commercial rainbow trout hatchery with a low-adhesion polymer created a smooth surface facilitating the dislodgement and elimination of the cyst stage of I. multifilis by natural flushing or brushing. The physical removal of the cyst stage alone, through the use of a mechanical device or substrate detrivorous/algae feeder as a biological control agent, significantly reduced the propagation of I. multifiliis to a low level of infection without the need to deploy an additional chemical treatment. These studies demonstrate that the cyst is a key stage in the dynamics of I. multifiliis infection and its removal from the fish culture systems could constitute an effective and simple mean of managing I. multifiliis infections. The third experimental chapter explores the utilisation of molecular marker to characterise different isolates of I. multifiliis. The results highlight the unsuitability of the rDNA region (ITS-1 and ITS-2) and the strong potential of the mtDNA (COI) as molecular markers to discriminate isolates of I. multifiliis from distant geographical locations. It is suggested that genetic “barcoding” using mtDNA is the most effective method to identify I. multifiliis isolates. Importantly, genetic “barcoding” could allow associating I. multifiliis strains or geographical isolates with particular properties as regards their ecophysiology, pathogenicity and sensitivity to treatment, in order to improve the management of I. multifiliis infections according to the specific genetic isolate encountered. This research project demonstrates the efficacy of a range of new approaches against the propagation of I. multifiliis. Together, our findings contribute towards the development of a more effective and integrated system for managing I. multifliis infections in farm systems. The utilisation of physical methods and of environmentally friendly chemotherapeutants holds great potential for the control of I. multifiliis infections in organic fish production and in a broader context to any freshwater food fish farms affected by I. multifiliis.

Aim of this thesis was to use the ozonisation as a disinfection method for improving of water quality in the intensive fish farms using RAS (recirculating aquaculture system). The main assessed parameter was effect of ozone treatment on fish health and RAS functions and features. Two RAS were used in this study one with use of ozone treatment, one without ozone treatment (control system). There were cultured two different fish species in these two RAS - pikeperch (Sander lucioperca) and European catfish (Silurus glanis). The main reason for use of these two species is that they are perspective species for intensive aquaculture. In the RAS with ozone treatment, two different methods of ozone application were tested - periodical and continual application. The effect of ozone treatment on fish health and conditions was controlled regularly. Ozone treatment had positively affected the survival of both cultured species (pikeperch survival: with ozone = 77.0 % and without ozone = 67.2 %; European catfish: with ozone = 93.1 % and without ozone = 91.5 %). Ozone treatment also positively affected the water chemistry. The greatest difference was observed in CHSKMn: with ozone = 6.4?1.2 mg.l; without ozone = 10.7?1.6 mg.l. The same features were observed in suspended solids: with ozone = 4.3?2.8 mg.l-1; without ozone = 8.17?6.2 mg.l. Appearance of Ichthyophthirius multifiliis and bacterial infection were not affected by ozone treatment. The main result of this thesis and this design of experiment is that ozone treatment had a positive impact on water chemistry in observed RAS and it, however, didn't kill 100% of the fish pathogens.

Tento pokus je považován za pilotní projekt. Nikdo nezkoušel aplikovat pouze KPO do rybničního prostředí. Metodicky bylo postupováno v souladu s projektem Aquaexcel. Výzkum byl zaměřen na 6 modelových rybníků na území pokusnictví VÚRH ve Vodňanech. Zde byla vysazena obsádka K1, L1 a L2. Od 26.6.2013 do 1.8.2013 byly pravidelně odebírány vzorky ryb pro parazitologické vyšetření. 2 tyto rybníky byly označeny jako kontrolní a ve zbývajících 4 došlo k aplikaci KPO v koncentraci 1 mg×l-1.K aplikaci docházelo 1 x nebo 2 x denně.V období od 30.5. do 4.6.2013 zasáhly Jihočeský kraj povodně a dešťové srážky. Dešťová voda společně se splachy z okolí promíchala a zvířila vodní sloupec všech těchto rybníků. Tato událost byla důvodem zvýšeného výskytu parazitární prevalence v počátcích odběrů vzorků, v porovnání s pozdějšími odběry vzorků. Celková incidence parazitů byla však velmi nízká. Líni byli zasaženi více parazitárními druhy, ale v mnohem menším počtu než v případě kaprů. Kaprům byla věnována největší pozornost a tedy i grafické a statistické vyhodnocení. U kaprů byli nalezeni parazité Trichodina sp. na žábrách a kůži, Ichthyophthirius multifiliis na žábrách a kůži, Apiosoma sp. na kůži, Gyrodactylus sp. na kůži. Po aplikaci KPO došlo k statisticky významnému snížení (P<0,01) pouze u Trichodina sp. na žábrách při porovnání kontrolních rybníků s rybníky. Rybniční prostředí se svými fyzikálně chemickými vlastnostmi liší od vody použité v průtočných systémech či v RAS. Pokus nevyšel dle očekávání, ale vzhled k tomu, že byl pilotní, tak nelze označit jako neúspěšný. Tento výzkum dává prostor pro další studie zaměřené na danou tématiku.