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.