ETosis in tambaqui Colossoma macropomum: A programmed cell death pathway and approach of leukocytes immune response.

Tambaqui Colossoma macropomum is the most cultivated native fish in South America and Aeromonas hydrophila is one of the main bacteria infecting tropical fish. Despite the economic importance of this round fish, to date, there has been a paucity of investigations into haematological changes in tambaqui. In this study, detailed blood analyses (0 h, 6 h, 24 h, 7 d and 14 d) following intraperitoneal challenge with A. hydrophila were performed. After analysing the results, there was a suspicion of a novel cell death mechanism via extracellular traps (ETosis) in tambaqui. The search for ETosis was based on differential interference contrast (DIC) microscopy and scanning electron microscopy (SEM) assays through application of an adapted protocol applying co-incubation of leukocytes with A. hydrophila. The cells were investigated at: 0 h (control), 4 h and 7 h after incubation. The complete haemogram profile showed an uncommon severe leukopenia in early phases of infection (6 h, p < 0.001 and ≤ 0.05), due to significant decreases in the three main leukocytes: lymphocytes (6 h, p ≤ 0.001), monocytes (6 h, p ≤ 0.05) and neutrophils (6 h and 24 h, p ≤ 0.01 and p ≤ 0.05). Leucocytosis and lymphocytosis (p ≤ 0.01) were ascertained only 7 days post-infection. Through DIC and SEM, we discovered that leukocyte suicide exposed the nuclear contents between 4 and 7 h after stimuli with bacteria. The leukogram profile associated with DIC and SEM analyses suggested that tambaqui leukocytes underwent a programmed death (ETosis) in order to expose chromatin and granule proteins as a trap to bind and then kill bacteria; thus, preventing A. hydrophila from spreading and resulting in leukopenia during the early phase of bacterial infection. In this paper, we presume that ETosis is one of the last resources for tambaqui to contain the infection, and after this leukocyte strategy, a high number of phagocytic cells are produced and released into the peripheral circulation.

Patterns of the innate immune response in tambaqui Colossoma macropomum: Modulation of gene expression in haemorrhagic septicaemia caused by Aeromonas hydrophila.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is not well understood. In this study we established some patterns of innate immunity for the species via two experiments. Both studies evaluated the fish in the absence (intraperitoneal saline) or presence (intraperitoneal, 3 x 107 CFU/mL of Aeromonas hydrophila at 0.1 mL/10 g of living weight) of infection at 5 points over time-course of 14 days (0 h, 6 h, 24 h, 7 d, 14 d). In the first experiment, the partial gene sequences and gene expression of IL-1ß, IRAK-1, C3, C4, lysozyme, IL-10, HSP70 and ß-actin were determined in the main secondary lymphoid organs of fish: the spleen and head kidney. The second study was performed to analyse the alternative complement pathway ACH50 in serum to support the elucidation of C3 gene expression. Results of the gene expression assays showed a tendency towards up-regulation of immune genes in infected fish in early phases of infection (mostly around 6 h and 24 h) and in the chronic phase (7 d and 14 d), with the exception of HSP70 which showed a down-regulation in infected fish. Our results also suggested that lysozyme was evolved in both pro- and anti-inflammatory activities. For genes of the complement system, it was demonstrated that C4 regulation followed the tendency of pro-inflammatory genes. However, the C3 gene was, surprisingly, not expressed in most fish and this corroborated with the results of the complement system activity in serum that also did not show activity in most fish. The possible reasons for the regulation of gene expression and association with fish disease are addressed in this paper.

Motile Aeromonas septicemia in tambaqui Colossoma macropomum: Pathogenicity, lethality and new insights for control and disinfection in aquaculture.

Tambaqui Colossoma macropomum is the most produced native fish in South America. Besides the lack of knowledge regarding bacteria-stricken diseases, the unappropriated using of off-label therapies are common. In this study, Aeromonas hydrophila pathogenicity for tambaqui was first established by Koch's Postulate. Lethal doses (LD) were settled for investigation of clinical signs and mortality. The antimicrobial activities were investigated by disk-diffusion test against 11 antibiotics and by broth microdilution methods against 3 antibiotics, 7 disinfectants and 11 herbal medicines. LD experiment showed up to 80% of fish mortality, skin darkness, ulcers, hemorrhage, lethargy and hypo/anorexia in all groups, with exception of control. The LD10,50,90 and 99 were established in 4.1 × 107, 8.8 × 107, 1.9 × 108 e 3.6 × 108 CFU/mL, respectively. Ceftriaxone, florfenicol, oxytetracycline and thiamphenicol were considered promising against A. hydrophila. All herbal medicines were classified as bactericides, but clove Eugenia caryophyllata and cinnamon Cinnamomum zeylanicum displayed strongest activities. Among disinfectants, malachite green was the only that did not present acceptable values, discouraging its use. In conclusion, Koch's postulate was fulfilled and tambaqui entered to the vast list of A. hydrophila hosts and promising results of chemical substances were provided, contributing to motile Aeromonas septicemia (MAS) control.

Trichodina heterodentata (Ciliophora) infestation on Prochilodus lineatus larvae: a host-parasite relationship study.

Prochilodus lineatus is a freshwater fish species found in South America. It is common in aquaculture, but few studies regarding diseases of this fish have been performed. This study presents data of the occurrence of Trichodina heterodentata Duncan, 1977, as well as the pathological alterations detected by light and scanning electron microscopy (SEM). Twenty 20-day-old larvae were harvested from an earth pond and examined. Larvae showed erratic swimming on the pond edges and some had a whitish tegument. Larval smears were either impregnated with silver nitrate or stained with Giemsa stain to observe the taxonomic features of the ciliates. Five larvae were fixed in formalin solution for histopathological analysis, and another five specimens were fixed in glutaraldehyde for SEM. All larvae were diagnosed with a severe infestation by trichodinid T. heterodentata. Histological sections showed discrete hyperplasia of the gill filaments with subepithelial oedema of the secondary lamellae. In the SEM, suction areas were observed on the skin, gill and eye; corrosion and ulceration of the fins were associated with the bacterial presence of cocci on the lesions. This is the first report of T. heterodentata in P. lineatus that is responsible for an acute disease that culminates in larval mortality.

Enterogyrus spp. (Monogenea: Ancyrocephalinae) and Aeromonas jandaei co-infection associated with high mortality following transport stress in cultured Nile tilapia.

Monogenean infection of the internal organs is extremely rare when compared to external infections. This study describes mass mortality of Nile tilapia (Oreochromis niloticus L.) originating from co-infection with Enterogyrus spp. and Aeromonas jandaei following transport stress. The first fish deaths occurred on day 1 post-transport, while cumulative mortality reached approximately 90% by day 10 post-stocking. An atypical amount of pale (whitish) faeces floating on the surface of the water as well as typical clinical signs of motile Aeromonas septicemia, were reported. Adult monogeneans and countless eggs of monogeneans were found in the stomachs and the intestines of both moribund and dead fish, respectively. Two strains of A. jandaei were isolated from the kidneys. Scanning electron microscope microphotographs of the stomach revealed the presence of numerous monogeneans penetrating deep into the gastric tissue, and diffuse lesions filled with bacilliform bacteria. Histopathological examination showed multifocal eosinophilic infiltrate, gastric gland and epithelial necrosis with sloughed necrotic debris in the lumen. This is the first report of co-infection by Enterogyrus spp. and A. jandaei in Nile tilapia and the first report of Enterogyrus coronatus, Enterogyrus foratus, and Enterogyrus malbergi parasitizing tilapia in Brazil. These findings indicate that synergic co-infection by Monogenean stomach parasites (E. coronatus, E. foratus, and E. malbergi) and A. jandaei may induce high mortalities in tilapia following transport stress.

First report of Trichodinella and new geographical records of trichodinids in Nile tilapia (Oreochromis niloticus) farmed in Brazil.

Massive occurrence of trichodinids is frequently accompanied by serious disease in fish farms. In this study, trichodinid species from the gills and skin of Nile tilapia (Oreochromis niloticus) farmed in the central-western region of Brazil (state of Goiás) were morphologically characterized. Dried slides were prepared from the parasites and were impregnated with silver nitrate (2%). Morphometric characteristics were determined and schematic drawings of the denticles were made using photomicrographs produced from the slides. Seven species of trichodinid ectoparasites (Protozoa: Ciliophora: Trichodinidae) were found parasitizing the gills: four of the genus Trichodina Ehrenberg, 1838; one of Tripartiella Lom, 1959; one of Paratrichodina Lom, 1963; and one of Trichodinella Srámek-Husek, 1953. On the body surface, three specimens of the genus Trichodina were identified. This study presents new geographical records of trichodinids in Brazil, thus confirming that Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana and Tripartiella orthodens are widely distributed worldwide. Additionally, the first record of the genus Trichodinella in Brazil is presented.

Trichodina modesta: an exotic ciliate in the Neotropical region parasitizing an unusual host.

In this study, an important ornamental fish, Betta splendens (Osphronemidae), from three different Brazilian states was examined for parasitic infestations. Smears with parasites were impregnated with silver nitrate or stained using Giemsa for taxonomic evaluation. A disc-shaped trichodinid with a body diameter of 39.7 ± 3.3 µm, adhesive disc diameter of 32.9 ± 3.1 µm and denticulate ring diameter of 19.5 ± 2.0 µm was found. The morphological characteristics resembled those of Trichodina modesta Lom, 1970, a species that shows clear host specificity for Cypriniformes. Until now, its occurrence was restricted to the Eurasian region. In the present study, a new host for T. modesta is reported and therefore the first occurrence of this species in the Americas. The parasite was possibly introduced into the Neotropical region through the exotic fish trade, especially of Cypriniformes used by aquarists. The distribution of this ciliate is discussed and a checklist of localities and hosts for the species is provided.

First report of Trichodinella and new geographical records of trichodinids in Nile tilapia (Oreochromis niloticus) farmed in Brazil / Primeiro relato de Trichodinella e novos registros geográficos de tricodinídeos em tilapia do Nilo (Oreochromis niloticus) cultivada no Brasil

Abstract Massive occurrence of trichodinids is frequently accompanied by serious disease in fish farms. In this study, trichodinid species from the gills and skin of Nile tilapia (Oreochromis niloticus) farmed in the central-western region of Brazil (state of Goiás) were morphologically characterized. Dried slides were prepared from the parasites and were impregnated with silver nitrate (2%). Morphometric characteristics were determined and schematic drawings of the denticles were made using photomicrographs produced from the slides. Seven species of trichodinid ectoparasites (Protozoa: Ciliophora: Trichodinidae) were found parasitizing the gills: four of the genus Trichodina Ehrenberg, 1838; one of Tripartiella Lom, 1959; one of Paratrichodina Lom, 1963; and one of Trichodinella Ṧrámek-Huṧek, 1953. On the body surface, three specimens of the genus Trichodina were identified. This study presents new geographical records of trichodinids in Brazil, thus confirming that Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana and Tripartiella orthodens are widely distributed worldwide. Additionally, the first record of the genus Trichodinella in Brazil is presented.

Resumo O parasitismo intenso por tricodinídeos está frequentemente relacionado à doença grave em fazendas de peixes. Neste estudo, espécies de tricodinídeos das brânquias e da pele de tilápias do Nilo (Oreochromis niloticus) cultivadas na região centro-oeste do Brasil (estado de Goiás) foram caracterizadas morfologicamente. As lâminas secas foram preparadas a partir dos parasitas e impregnadas com nitrato de prata (2%). As características morfométricas foram determinadas e desenhos esquemáticos dos dentículos foram confeccionados com fotomicrografias produzidas a partir das lâminas. Sete espécies de ectoparasitos tricodinídeos (Protozoa: Ciliophora: Trichodinidae) foram encontradas parasitando as brânquias: quatro do gênero Trichodina Ehrenberg, 1838; um de Tripartiella Lom, 1959; um de Paratrichodina Lom, 1963; e um de Trichodinella Ṧrámek-Huṧek, 1953. Na superfície do corpo, três espécimes do gênero Trichodina foram identificados. Este estudo apresenta novos registros geográficos de tricodinídeos no Brasil, confirmando que Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana e Tripartiella orthodens estão amplamente distribuídas mundialmente. Adicionalmente, é apresentado o primeiro registro do gênero Trichodinella no Brasil.

Trichodina modesta: an exotic ciliate in the Neotropical region parasitizing an unusual host / Trichodina modesta: exótico ciliado na região Neotropical parasitando hospedeiro não usual

In this study, an important ornamental fish, Betta splendens (Osphronemidae), from three different Brazilian states was examined for parasitic infestations. Smears with parasites were impregnated with silver nitrate or stained using Giemsa for taxonomic evaluation. A disc-shaped trichodinid with a body diameter of 39.7 ± 3.3 µm, adhesive disc diameter of 32.9 ± 3.1 µm and denticulate ring diameter of 19.5 ± 2.0 µm was found. The morphological characteristics resembled those of Trichodina modesta Lom, 1970, a species that shows clear host specificity for Cypriniformes. Until now, its occurrence was restricted to the Eurasian region. In the present study, a new host for T. modesta is reported and therefore the first occurrence of this species in the Americas. The parasite was possibly introduced into the Neotropical region through the exotic fish trade, especially of Cypriniformes used by aquarists. The distribution of this ciliate is discussed and a checklist of localities and hosts for the species is provided.

Neste estudo, o peixe ornamental Betta splendens (Osphronemidae) foi examinado a partir de três diferentes Estados brasileiros para infestações parasitárias. Esfregaços contendo parasitos foram impregnados com nitrato de prata ou corados com Giemsa para avaliação taxonômica. Foi observado o tricodinídeo em forma de disco com 39,7 ± 3,3 µm de diâmetro do corpo, disco adesivo com 32,9 ± 3,1 µm e anel denticulado 19,5 ± 2,0 µm de diâmetro. As características morfológicas foram semelhantes à Trichodina modesta Lom, 1970, uma espécie que apresenta especificidade de hospedeiro por peixes Cypriniformes com ocorrência na Região Euro-asiática. No presente estudo, um novo hospedeiro para T. modesta é reportado, assim como a primeira ocorrência da espécie na América. O parasito tem sido possivelmente introduzido na região Neotropical com o comércio de peixes exóticos, principalmente Cypriniformes usados por aquariofilistas. A dispersão do ciliado é discutida e um “checklist” de localidades e hospedeiros apresentado.