Academic literature on the topic 'Fish histopathology'

Abstract Koi fish (Cyprinus carpio koi) is one of the ornamental fish are preferred. One of the obstalces in the koi fish is a disease caused by infection myxobollus. This research to determine the effect of the infection myxobollus by oral in blood picture and histopathology. Haematology and histopathology is an indicator of the health of the fish to diagnose disease and to determine changes in the fish tissue. The research was conducted in Politeknik Kelautan dan Perikanan, dan Balai Karantina Ikan Kelas I, Juanda Surabaya from October to December 2014. This study uses the 5 treatments and 5 replications with different doses of infection (0,60,80,100 and 120 spora/fish). Data were analyzed with Annova haematology, followed by Duncan 5%. While histopathology were analyzed by semiquantitative scoring mothod used to determine the degree of histology Kluschal Wallis. The result showed that the haematology has significant result, white histopathology signify the presence of significant damage.

The present study proposes to characterize the parasites isolated during the initial phase of production in fish farms located in Mato Grosso do Sul in the central-western region of Brazil, using histopathology analysis and molecular techniques. A total of 340 hybrid surubim fish (Pseudoplatystoma reticulatum × P. corruscans) from four farms were examined during the co-feeding phase. Histopathology analysis showed that 10.9% (n = 37) of the fish were infected with parasites. Branchitis, lifting epithelium, hypertrophy of epithelial cells, lamellar fusion, aneurisms and infection in the bone tissue of the gill filament was observed. The parasite species was determined by amplification of the 18S rRNA gene followed by sequencing. The phylogenetic analysis of nucleotide sequences indicates a close relationship (99.6%) with Henneguya pseudoplatystoma reported to be infecting the hybrid Pseudoplatystoma. This study demonstrates the occurrence of H. pseudoplatystoma in hybrid surubim (P. reticulatum × P. corruscans) during the co-feeding phase in fish farms in Mato Grosso do Sul. Also, molecular techniques provide a faster and sensitive method to identify fish parasites, and may assist in the development of new management techniques aimed at improving the sanitary conditions contributing to the reduction of mortality rates in these animals.

Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are putative viral cardiac diseases of Atlantic salmon. This study examined the levels and correlated the serum enzymes creatine kinase (CK) and lactate dehydrogenase (LDH) to the histopathology of clinical outbreaks of HSMI and chronic CMS in farmed Atlantic salmon. A total of 75 fish from 3 different HSMI outbreaks, 30 chronic CMS fish, and 68 fish from 3 nondiseased fish groups were used as the study population (N=173). Serum CK and LDH levels correlated significantly with the total inflammation and total necrosis scores for HSMI fish (P=0.001). However, no correlation was identified for enzyme levels and histopathology scores for chronic CMS fish. The significantly increased CK and LDH levels and their positive correlations to histopathology differentiate HSMI from CMS clinically suggesting the potential use of enzymes for screening for HSMI is promising.

Mercury (Hg) which is contained in aquatic ecosystem can enter and be accumulated to organism’s body, like on petek fish (Leiognathus equulus). The research aimed to see mercury concentration in aquatic ecosystem, to see mercury concentration in organ of petek fish by AAS (Atomic Absorption Spectrophotometer) and its histopathology response in organs of petek fish. The research was done at Ancol, Jakarta Bay on October-December 2004. There are 16 samples of water and fish from 3 stations.The analysis result of water quality is compared by standard quality of sea water for sea organism life (Kepmen LH No. 51, 2004), mercu ry concentration in fish organ is compared by maximum mercury concentration in fish body by classification of Palar (2004). Ancol water quality is still on normal condition. Mercury concentration in water and petek fish organs had low concentration. Gill and lever contaminated by mercuryis only in station 1 fish, but it is still on normal concentration. Histopathology of petek fish gill is not abnormal, while the lever is necrosis. Water and petek fish is not a good indicator to detect mercury in aquatic ecosystem.Keywords: mercury, petek fish, water, lever, gill, histopathology, accumulation,indicator

Pesticides are pollutants that are found in rice fields and rivers. Pesticides that are often used by farmers in Indonesia in eradicating insects are organophosphate insecticides, where they can eradicate insects that are very toxic to fish due to strong neurotoxic substances that inhibit AchE (Acetylcholinesterase) activity. The research aims to examine the effect of organophosphate pesticides on hematology and histopathology of hematopoietic organs in silver rasbora fish. The research method used is an experimental method with a CRD. The parameters observed were hematology and histopathology hematopoietic organ (liver and kidney). The results of this study showed a hematological change in silver rasbora fish where there was a decrease in total erythrocytes (0,59±0,004) and hemoglobin (2,5±0,1) while total leukocytes increased (245,35±15,78). Also, there are differential changes in leukocytes, namely an increased in the number of monocytes (5±1) and neutrophils (24±3), but lymphocytes have decreased in number (72±1). The Histopathology of the fish liver also can found in this research, those damages that are found are erythrocyte infiltration, necrosis picnosis, and karyolysis. Histopathology of fish kidney also can found cloudy sweling, necrosis karyolysis and tubular necrosis.

Histopathology studies of metal nanoparticles (NPs) compared to traditional forms of metal in fish are scarce. Additionally, it is unclear whether metal nanoparticles cause greater or different pathologies compared to other forms of metal. The current study aimed to assess the pathological effects of Cu-NPs and TiO2 NPs on rainbow trout via various routes of exposure and, where appropriate, to compare them to either the equivalent dissolved metal salts or bulk powder forms. The first experiment showed that waterborne exposure to Cu-NPs and CuSO4 caused similar types of organ pathologies and alteration in the spleen content, however there were some material-type effects in the incidence injuries; with Cu-NPs in some organs by causing more injury in the intestine, liver, and brain when compared to effects caused by the equivalent concentration of CuSO4. Lowering water pH did have an effect on the toxicity of Cu-NPs and dissolved Cu in trout, and the results illustrated that both Cu treatments are more toxic at pH 5 than pH 7 by causing more physiological and pathological changes, although both CuSO4 and Cu-NP treatments showed similar types of organ lesions. Waterborne exposure to TiO2 NPs and bulk forms of TiO2 showed similar types of organ pathologies and alteration in the spleen contents, but there was a material-type effect in some organs (more injury with the bulk treatment than the NP form). After 96 h following intravenous injections of bulk or TiO2 NPs in trout, organs showed similar types of pathologies; except the spleen and kidney which showed a material-type effects (more injury with NPs than the bulk forms). This could be attributed to the highest Ti accumulation from the TiO2 NP treatment in the kidney and spleens, or to the role of these organs in filtrating the circulating blood. Overall, this thesis demonstrates that metal-NPs produced similar types of organ pathologies to traditional forms of metals through different routes of exposure, but there were some material-type effects on the incidence of injuries in some organs. The results have also added some understanding on the fate, and effects of NPs by identifying the target organs involved. Some of the nano-specific effects may need to be given extra consideration in environmental and human health risk assessments.

Fewer than 200 fish species are found in freshwater habitats in Australia, of which 144 are confined exclusively to freshwater. At least 22 species of exotic freshwater fish have been introduced into Australia, and 19 of these have established self-sustaining populations. However, the parasite fauna of both native and exotic freshwater fishes in Australia is poorly known. This is particularly the case in the south-west of Western Australia, where there have been no previous comprehensive studies of the parasites of 14 native species and nine or more exotic species of fish found in freshwater habitats. This study represents a survey of the parasites of freshwater fishes in the South West Coast Drainage Division and reports 44 putative species of parasites in 1429 individual fishes of 18 different species (12 native and six exotic) from 29 locations. Parasites were found in 327 (22.88%) fishes, and of the infected fishes, 200 (61.16%) were infected with only one species of parasite and 127 (38.84%) were infected with two or more species of parasites. For helminth and arthropod parasites, which were more comprehensively surveyed than protozoan and myxozoans, I found 37 species compared to 77 species found in a recent study of fishes from the East Coast Drainage Division. The present study demonstrated that parasitic infection was significantly more common in native fish species (mean prevalence of infection with any species of parasite = 0.36 ± 0.09) than in exotic fish species (0.01 ± 0.12). Parasites were found in all native fish species, but in only two exotic fish species that were examined. Parasite regional and component community diversity were estimated by species richness (the number of species, S) and by an index of taxonomic diversity (HT). Both parasite species richness and parasite taxonomic diversity were significantly greater in native fish species (mean S = 10.5 ± 2.3; mean HT = 1.19 ± 0.14) than in exotic fish species (mean S = 1.6 ± 3.3; mean HT = 0.27 ± 0.20). These relationships were consistent over all geographic locations that were sampled. The reduced parasite load of exotic species compared to native species has been previous reported across a wide range of taxa. It is thought to arise partly because founding populations of hosts have a low probability of harbouring the species’ total parasite fauna, and partly because parasites that infect introduced exotic species may not be able to maintain their life cycle in the new environment. It has been suggested that a reduced parasite load increases the competitive ability of exotic species compared to native species (the parasite release hypothesis) and this may partly explain the abundance and apparent competitive success of exotic over native species of freshwater fish in the South West Coast Drainage Division. For native species of fish, there were major differences among species in both prevalence of parasitic infection and parasite community diversity, but this variation was not related to fish size, whether the fish were primarily freshwater or primarily estuarine, or whether they were primarily demersal or pelagic. In this study, I report two new parasites in south western Australian waters. Both are copepod parasites; Lernaea cyprinacea and a new species of Dermoergasilus. The Dermoergasilus appears to be native to the south-west of Western Australia and has been described as Dermoergasilus westernensis. It differs from previously described species in the genus principally by the armature of the legs. This new species was found on the gills of freshwater cobbler, Tandanus bostocki and western minnow, Galaxias occidentalis in two different river systems. Lernaea cyprinacea is an introduced parasitic copepod found on the skin and gills of freshwater fishes in many areas of the world. The parasite has not previously been reported in Western Australia. We found infestations of L. cyprinacea on four native fish species (G. occidentalis; Edelia vittata; Bostockia porosa; T. bostocki) and three introduced fish species (Carassius auratus; Gambusia holbrooki; Phalloceros caudimaculatus) at two localities in the Canning River, in the south-west of Western Australia. The parasite has the potential to have serious pathogenic effects on native fish species, although it appears to be currently localised to a small section of the Canning River. Over all localities from which fishes were sampled in the present study, the proportion of native freshwater fishes with parasitic infections and the component community diversity of the parasite fauna of native fishes were both negatively related to habitat disturbance, in particular to a suite of factors (river regulation, loss of riparian vegetation, eutrophication and presence of exotic fish species) that indicate increased human usage of the river and surrounding environment. The reduced parasite load and diversity in native fishes from south-west rivers with greater human usage was due principally to the loss of a number of species of trematode, cestode and nematode endoparasites which use fishes as intermediate hosts. Other studies have also found that endoparasites with complex life cycles are most likely to be adversely affected by environmental changes, presumably because any environmental changes which impact on either free-living parasite stages or on any of the hosts in the complex train of parasite transmission will reduce parasite population size and may cause local extinction of the parasite species. The most heavily infected species of native freshwater fish in the South West Coast Drainage Division was T. bostocki with 96% of all individuals containing at least one species of parasite. As with most freshwater fishes of south-west Australia, T. bostocki is limited in its distribution to waterways with relatively low salinity. The degree of parasitism and histopathology of internal and external organs in T. bostocki from the Blackwood River was examined over a period of rapid, seasonal changes in water salinity. As salinity increased, the infracommunity richness and prevalence of ectoparasites on the skin of fishes decreased, while the infracommunity richness and prevalence of endoparasites increased. This was associated with a decrease in histopathological lesion scores in the skin and an increase in histopathological lesion scores in internal organs, particularly the intestine. I hypothesise that the seasonal spike in salinity had two contrasting effects on parasitic infections of T. bostocki. Firstly, it increased the mortality rate of parasites directly exposed to water, leading to a decrease in ectoparasitic infection and associated pathology. Secondly, it suppressed immune function in fish, leading to a decreased mortality rate of parasites not directly exposed to water and a more severe pathological response to endoparasitism.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol)
Ichthyophonus hoferi is a highly pathogenic histozoic parasite that has low host specificity capable of producing mass mortalities of epizootic proportions in marine commercial fish populations. Currently in Southern Africa, I. hoferi has been reported from flathead mullet (Mugil cephalus) from the Kowie lagoon and from multiple species on exhibit at the Two Oceans Aquarium. Since epizootiologists rely on accurate assessments of prevalence to establish patterns of morbidity and mortality within populations, using the most accurate diagnostic techniques for accurate assessments of infection is imperative. Currently, several diagnostic techniques have been employed to detect I. hoferi in infected fish hosts. These include macroscopic examination of tissues, microscopic examinations of wet-mount squash preparations of tissue, histological examination of tissue sections, in vitro culture of tissue explants, the polymerase chain reaction (PCR) using I. hoferi-specific primers and real-time quantitative PCR (qPCR) using I. hoferi-specific primers and a hydrolysis probe.

A toxicidade da formulação comercial contendo o praguicida piretróide deltametrina foi testada nas espécies de peixe Danio rerio, Hyphessobrycon bifasciatus, Geophagus brasiliensis e Oreochromis niloticus por meio de testes de toxicidade aguda, avaliação de alterações comportamentais e análises anatomopatológicas do fígado e das brânquias. A espécie D. rerio apresentou CL50;48h igual a 0,078µg L-1, a espécie H. bifasciatus apresentou CL50;48h igual a 0,082µg L-1, a espécie G. brasiliensis apresentou CL50;48h igual a 0,594µg L-1 e O. niloticus apresentou CL50;48h igual a 0,954µg L-1. As análises de comportamento realizadas com as espécies D. rerio e H. bifasciatus mostrou ser a segunda mais sensível ao praguicida, revelando alterações significativas nas menores doses de exposição. As análises anatomopatológicas realizadas com a espécie G. brasiliensis quando exposta à concentrações subagudas do praguicida mostrou vacuolização e degeneração nuclear hepática e nas brânquias a ocorrência de hiperplasia lamelar e fusão nas lamelas secundárias que aumentaram em função da concentração do praguicida e do tempo de exposição. Os resultados demonstraram elevada toxicidade do praguicida para todas as espécies testadas.
The toxicity of the commercial formulation containing the pesticide pirethroid deltamethrin was tested in the species of fish Danio rerio, Hyphessobrycon bifasciatus, Geophagus brasiliensis and Oreochromis niloticus through tests of acute toxicity, evaluation of behavioral alterations and anatomopathological analysis of the liver and of the gills. The species D. rerio presented LC50;48h equals to 0,078µg L-1, the species H. bifasciatus presented LC50;48h equals to 0,082µg L-1, the species G. brasiliensis presented LC50;48h equals to 0,594µg L- 1 and O. niloticus presented LC50;48h equals to 0,954µg L-1. The analysis of behavior accomplished with the species D. rerio and H. bifasciatus showed that the second one was more sensitive to the pesticide, and revealed significant alterations in the exhibition of fewer doses. The anatomopathological analysis accomplished with the species G. brasiliensis, when exposed to underacute concentrations of the pesticide, showed vacuolization and hepatical nuclear degeneration and the occurrence of lamellar hyperplasia in the gills plus fusion in the secondary lamellas which were increased due to the concentration of the pesticide and of the time of exposition. The results demonstrated high toxicity of the pesticide for all of the tested species.

As cidades e as atividades industriais e agrícolas geram uma infinidade de substâncias, muitas vezes desconhecidas, que quando lançadas nos corpos de água receptores podem por em risco a biota local e até mesmo a saúde humana. O presente trabalho avaliou o estado de degradação ambiental nas bacias dos rios Piracicaba, Mogi Guaçu (ambas no estado de São Paulo) e Tibagi (estado do Paraná), tendo como ferramentas os testes de toxicidade de sedimentos com peixes, em laboratório e in situ, análises histopatológicas dos animais expostos e análises físicas e químicas da água e do sedimento. Foram realizadas, ainda, análises das alterações histológicas em peixes expostos ao efluente final de indústria de papel e celulose, atividade presente nas três bacias estudadas. Os organismos-teste selecionados foram a espécie nativa de peixe Serrapinnus notomelas (Characiformes, Characidae) e a espécie padronizada, exótica, Danio rerio (Cypriniformes, Cyprinidae). As preparações histológicas foram realizadas com os peixes inteiros, possibilitando a análise dos órgãos-alvo brânquias, rim e fígado, em um mesmo corte. Nenhum organismo morreu após 96 horas de exposição às amostras de sedimento. No entanto, as análises histológicas revelaram alterações importantes, as quais foram sistematizadas para o cálculo do Índice de Alterações Histológicas (IAH) e do Valor Médio de Alterações (VMA), por órgão e por espécie, para cada localidade estudada. Os sedimentos provenientes das localidades de Sumaré (rio Atibaia) e de Piracicaba (rio Piracicaba) foram os mais tóxicos a ambas as espécies, de acordo com os índices calculados, seguidos pelos sedimentos de Campinas (rio Atibaia), Limeira (rio Jaguari) e Americana (rio Piracicaba). Os sedimentos da bacia do rio Mogi Guaçu foram menos tóxicos, mas aqueles provenientes das localidades Mogi Guaçu e Luiz Antônio (ambos a jusante de indústrias de papel e celulose) provocaram alterações renais e hepáticas significativas em D. rerio. Apesar de terem sido observadas alterações importantes nos peixes de ambas as espécies, expostas aos sedimentos da bacia do rio Tibagi, estas não foram tão intensas e freqüentes como aquelas observadas nos peixes expostos aos sedimentos das localidades das outras bacias hidrográficas. As análises físicas e químicas comprovaram a crítica situação de degradação do ambiente aquático na bacia do rio Piracicaba e alertam para os cuidados que deverão ser tomados com relação a esta e às bacias dos rios Mogi-Guaçu e Tibagi, cujos recursos estão sendo cada dia mais explorados. As graves alterações observadas nas brânquias, rim e fígado dos peixes expostos ao efluente de indústria de papel e celulose indicaram que o monitoramento da toxicidade desse tipo de efluente deve ser realizado por ferramentas mais sensíveis, tais como as análises histopatológicas em peixes.
Urban and industrial activities generate a large number of substances, frequently unknown, that if discharged into water body receptors can put at risk the whole aquatic biota, as well as human health. The present work evaluated the state of environmental degradation in the basins of rivers Piracicaba, Mogi Guaçu (both in São Paulo state) and Tibagi (Paraná state), using as tools the toxicity tests with sediments and fishes as test-organisms, for both laboratory and in situ evaluations, together with histopathological analyses of exposed animals and chemical and physical analysis of sediments and water. Also, histological alterations in fishes exposed to the final pulp-mill effluent were analyzed. The species selected as test-organisms were the native species of fish Serrapinnus notomelas (Characiformes, Characidae) and the standard exotic species Danio rerio (Cypriniformes, Cyprinidae). Hystological preparations were performed with whole fish specimens, what allowed the analysis of the target-organs: gills, kidney and liver in the same section. No fish mortality was found after 96 hours exposition to the sediment samples. Nevertheless histological analyses revealed important alterations, which were used to calculate the histological alteration index (IAH) and the mean value of alterations (VMA), for each organ and species, in each locality evaluated. Sediments coming from Sumaré (Atibaia river) and Piracicaba (Piracicaba river) were the most toxic to both species, according to the calculated index, followed by the sediments from Campinas (Atibaia river), Limeira (Jaguari river) and Americana (Piracicaba river). The sediments of Mogi Guaçu riber basin had a lower toxicity, but those coming from Mogi Guaçu and Luiz Antônio (both localities placed downstream pulp-mill plants) have caused significant alterations in kidney and liver of D. rerio. Although important alterations were found for both species of fish exposed to the sediments from Tibagi river basin, these were not as intense and frequent as those observed in the fishes exposed to the sediment from the other localities studied. Physical and chemical analysis corroborated the critical degree of environmental degradation of Piracicaba river basin and must be considered as an early warning for the measures that must be taken in relation to this and also to Mogi-Guaçu and Tibagi basins, where resources are increasingly being overexploited. The severe damage found in gills, kidney and liver of fishes exposed to the pulp-mill effluent suggest that monitoring this type of effluent must include a high sensibility tool, as the histopatological analysis on fish.

Orientador: Sarah Arana
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Atrazina é um herbicida muito usado em agricultura intensiva e encontrado com alta freqüência em recursos hídricos na região do Pantanal Mato-grossense. Assim, devido aos riscos que a atrazina pode trazer à ictiofauna da região, foi proposta deste trabalho determinar a CL50 da atrazina em alevinos de pacu (Piaractus mesopotamicus). A determinação da CL50 em 96 h em sistema estático, realizada em duplicata, foi conduzida em aquários de vidro com 8 peixes cada, de peso médio de 5,06±0,31g, avaliando-se as seguintes concentrações nominais de atrazina: 0; 13,2; 17,6; 22,0; 26,4; 30,8; 35,2; 39,6 mg L-1, realizando-se também análise comportamental e análise anatomopatológica. Experimento de intoxicação aguda foi realizado em duplicata, nas mesmas condições do anterior, com a concentração da CL50 obtida (28,58 mg L-1), sendo empregados 6 exemplares de peso médio 6,68±0,36g. Amostras hepáticas e mesonéfricas foram colhidas e processadas para análise de microscopia de luz (ML), realizando-se nestas amostras análise semi-quantitativa das alterações encontradas, e para análise de microscopia eletrônica de transmissão (MET). Quanto à avaliação comportamental, foram observados nos grupos tratados: o escurecimento da pigmentação da pele, várias alterações na intensidade do movimento, perda de equilíbrio e presença da ação de boquejamento. Na avaliação anatomopatológica, foram observadas nos grupos tratados: dilatação da região ventral, exoftalmia, protrusão labial, hiperemia no opérculo e em todas as nadadeiras, presença de ar e/ou água no estômago e ascite sanguinolenta. Quanto à análise histopatológica, o grupo controle apresentou a típica morfologia hepática e renal para a espécie. No grupo tratado, severas alterações histopatológicas foram observadas em ML, a mais significativa em fígado foi a presença de inclusões hialinas no citoplasma dos hepatócitos e em rim a degeneração do túbulo proximal. À MET demonstrou que a atrazina causou severas alterações de organelas membranosas, sugestivas de estresse oxidativo e peroxidação lipídica, sendo as alterações mais freqüentes no fígado: inclusão lipídica nuclear e citoplasmática, tumefação do retículo endoplasmático rugoso e de mitocôndrias, degeneração do canalículo biliar com redução na quantidade de microvilos, e em rim, no túbulo proximal (TP): vacúolos citoplasmáticos e aumento do espaço intercelular na porção basolateral, figuras de mielina, tumefação mitocondrial, e, raramente, degeneração do TP. Estas alterações são compatíveis com intoxicação química, demonstrando que estes órgãos são bons biomarcadores de contaminação aquática em peixes. E por fim, o valor da CL50 sugere que a atrazina é levemente tóxica para o pacu, porém pelos demais resultados observados se inferem que só analisar a mortalidade não é o suficiente para determinar o dano causado por agrotóxicos em peixes, assim, recomenda-se o emprego de vários biomarcadores, tais como: análises comportamentais, histopatológicas, bioquímicas, etc.
Abstract: Atrazine is a widely used herbicide in intensive agriculture and a frequent contaminant of waterways in the Pantanal region of Brazil. In view of the potential risks of atrazine to the ichthyofauna of this region, in this work we examined the LC50 of atrazine in pacu (Piaractus mesopotamicus) fingerlings. For this study, fish (5.06 ± 0.31g; mean±SD) were housed eight per glass aquarium and exposed to various concentrations of atrazine (0, 13.2, 17.6, 22.0, 26.4, 30.8, 35.2 and 39.6 mg L-1) for 96 h in static system and this experiment was performed in duplicate, after which the LC50 was determined. Changes in fish behavior and anatomopathological analysis were monitored throughout the experiment. The histopathological alterations caused by atrazine were examined in six fish (6.68 ± 0.36g; mean±SD) using the LC50 (28.58 mg L-1) calculated from the concentration-response curve obtained above. This experiment was done in 96h in static system and performed in duplicate. Liver and mesonephric samples were processed for light microscopy (LM), performing in these samples the semi-quantitative analysis of the changes found, and for transmission electron microscopy (TEM) analysis. Exposure to atrazine caused darkening of the skin, alterations in the intensity of movements, loss of balance and an increase in the frequency of gasping. Anatomopathological assessment revealed dilation of the ventral region, exophthalmia, lip protrusion, skin hyperemia in the opercular region and in all fins, the presence of air and/or water in the stomach, and bloody ascites after the herbicide exposition. The histopathological analysis revealed the typical hepatic and renal morphology for the specie, in control group. Several histopathological changes were observed in exposed fish, but the changes most significant were: in liver, the presence of hyaline inclusions in the cytoplasm of hepatocytes, and proximal tubule (PT) degeneration in the kidney. The ultrastructure showed that the atrazine caused several membrane alterations suggestive of oxidative stress and lipid peroxidation. The most frequent in liver, TEM findings, were: nuclear and cytoplasmic lipid inclusions, swollen rough endoplasmic reticulum and mitochondria, degeneration in the bile canaliculi with a loss in the number of microvilli, and for kidney: an increase in the intercellular space of the basolateral region, myelin figures, swollen mitochondria, and, rarely, degeneration of PT. These lesions are consistent with chemical intoxication and indicate that the liver and kidney are good biomarkers of aquatic contamination in pacu. And, finally, the LC50 value suggests that atrazine is not highly toxic to pacu, however, use of mortality index in acute toxicity as the sole marker in ecotoxicological assays is inadequate and should be completed with other biomarkers such as behavioral, histopathology, biochemistry analyses, etc.
Mestrado
Histologia
Mestre em Biologia Celular e Estrutural

Streptococcus agalactiae is a pathogen of multiple hosts. The bacterium, an aetiological agent of septicaemia and meningo-encephalitis in freshwater and saltwater fish species, is considered a major threat to the aquaculture industry, particularly for tilapia. Cattle and humans are however the main known reservoirs for S. agalactiae. In humans, the bacterium (commonly referred to as Group B Streptococcus or GBS) is a member of the commensal microflora of the intestinal and genito-urinary tracts, but it is also a major cause of neonatal invasive disease and an emerging pathogen in adults. In cattle, S. agalactiae is a well-recognized causative agent of mastitis. Numerous studies focusing on S. agalactiae from human and bovine origins have provided insight into the population structure of the bacterium, as well as the genome content and pathogenic mechanisms through identification of virulence determinants. Concerning S. agalactiae from aquatic origins, scientific information mainly focused on case reporting and/or experimental challenges, with a limited or absence of information in terms of pathogenesis, virulence determinants and genotypes of the strains involved. The objective of this study was to enhance our understanding of the molecular epidemiology, host-adaptation and pathogenicity of S. agalactiae in aquatic species, with particular emphasis on tilapia. Firstly, a collection of 33 piscine, amphibian and sea mammal isolates originating from several countries and continents was assembled, with the aim of exploring the population structure and potential host specificity of aquatic S. agalactiae. Isolates were characterised using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and a standardised 3-set genotyping system comprising molecular serotypes, surface protein gene profiles and mobile genetic element profiles. Two major subpopulations were identified in fish. The first subpopulation consisted of non-haemolytic isolates that belonged to sequence type (ST) 260 or 261, which are STs that have been reported only from teleosts. These isolates exhibited a low level of genetic diversity by PFGE and clustered with other STs that have been reported only in fish. Another common feature was the absence of all surface protein genes or mobile genetic elements targeted as part of the 3-set genotyping and that are usually found in human or bovine isolates. The second subpopulation consisted of β-haemolytic isolates recovered from fish, frogs and sea mammals, and that exhibited medium to high genetic diversity by PFGE. STs identified among these isolates have previously been identified from strains associated with asymptomatic carriage and invasive disease in humans. The human pathogenic strain ST7 serotype Ia was detected in fish from Asia. Moreover, ST283 serotype III-4 and its novel single locus variant ST491 detected in fish from Southeast Asia shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. These observations suggested that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. STs found among the seal isolates (ST23) have also been reported from humans and numerous other host species, but never from teleosts. This work provided an excellent basis for exploration of the virulence of selected strains in experimental challenges. The virulence of two strains of S. agalactiae was experimentally investigated by intra-peritoneal infection of Nile tilapia (Oreochromis niloticus), using an isolate originally recovered from fish and belonging to ST260, and an isolate originating from a grey seal and belonging to ST23. The clinical signs, the in vivo distribution of viable bacteria and bacterial antigens, and the gross and histopathological lesions that developed during the time course of the infection were investigated. The ST260 strain was highly virulent, whereas no major clinical sign or mortalities occurred in the fish challenged with the ST23 strain. After injection, both strains however gained access to the bloodstream and viable bacteria were recovered from all organs under investigation. During the early stages of infection, bacteria were mostly found within the reticulo-endothelial system of the spleen and kidney. Thereafter, the ST260 demonstrated a particular tropism for the brain and the heart, but granulomatous inflammation and associated necrotic lesions were observed in all organs. ST23 was responsible for a mixed inflammatory response associated with the presence of bacteria in the choroid rete and in the pancreatic tissue only. After 7 days post-challenge and for both strain, the formation or containment of bacteria within granulomata or other encapsulated structures appeared to be a major component of the fish response. However, the load of viable bacteria remained high within organs of fish infected with ST260, suggesting that, unlike ST23, this strain is able to survive within macrophages and/or to evade the immune system of the fish. This work demonstrates that the lack of report of ST23 strains in fish is possibly not due to a lack of exposure but to a lack of virulence in this host. The two strains, which differ in prevalence and virulence in fish, provide an excellent basis to investigate genomic differences underlying the host-association of distinct S. agalactiae subpopulations. The genome of the ST260 strain used in challenge studies was sequenced. We therefore provided the first description for the genome sequence of a non-haemolytic S. agalactiae isolated from tilapia (strain STIR-CD-17) and that belongs by multi-locus sequence typing (MLST) to clonal complex (CC) 552, which corresponds to a presumptive fish-adapted subgroup of S. agalactiae. The genome was compared to 13 S. agalactiae genomes of human (n=7), bovine (n=2), fish (n=3) and unknown (n=1) origins. Phylogenetic analysis based on the core genome identified isolates of CC552 as the most diverged of all S. agalactiae studied. Conversely, genomes from β-haemolytic isolates of CC7 recovered from fish were found to cluster with human isolates of CC7, further supporting the possibility that some strains may represent a zoonotic or anthroponotic hazard. Comparative analysis of the accessory genome enabled the identification of a cluster of genes uniquely shared between CC7 and CC552, which encode proteins that may provide enhanced fitness in specific niches. Other genes identified were specific to STIR-CD-17 or to CC552 based on genomic comparisons; however the extension of this analysis through the PCR screening of a larger population of S. agalactiae suggested that some of these genes may occasionally be present in isolates belonging to CC7. Some of these genes, occurring in clusters, exhibited typical signatures of mobile genetic elements, suggesting their acquisition through horizontal gene transfer. It is not possible to date to determine whether these genes were acquired through intraspecies transfer or through interspecies transfer from the aquatic environment. Finally, general features of STIR-CD-17 highlighted a distinctive genome characterised by an absence of well conserved insertion sequences, an abundance of pseudogenes, a smaller genomic size than normally observed among human or bovine S. agalactiae, and an apparent loss of metabolic functions considered conserved within the bacterial species, indicating that the fish-adapted subgroup of isolates (CC552) has undergone niche restriction. Finally, genes encoding recognised virulence factors in human S. agalactiae were selected and their presence and structural conservation was evaluated within the genome of STIR-CD-17.

[ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales.
[CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals.
[EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections.
The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM).
Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035
TESIS

<em>Abstract.</em>—Runoff from dormant spray applications to orchards can contain the insecticides diazinon and esfenvalerate, which may affect the health of the threatened splittail <em>Pogonichthys macrolepidotus</em>. To examine the potential effects of these two insecticides, splittail larvae were exposed to diazinon, esfenvalerate, and a mixture of the two insecticides in static renewal 96-h acute toxicity tests. Surviving fish were transferred to clean water for an additional 14 d (18 d total). Mortality, morphological anomalies, histopathology, and growth determinations were made to assess lethal and sublethal effects. The combination of diazinon and esfenvalerate produced less than additive (independent) toxicity. Diazinon (singly and in combination with esfenvalerate) produced latent toxicity after the 96-h exposure, as demonstrated by reduced growth and increased spinal deformities. Metabolic dysfunction in the liver and inflammation of the pancreas were likely related to slower growth of diazinon-exposed fish. These symptoms were almost absent from esfenvalerate-exposed fish. The use of 14-d EC50 values and the measurement of biomarkers may more accurately describe the effects of 96-h shortterm exposure to these insecticides than traditional 96-h LC50 values.