RESUMO
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.
Assuntos
Caraciformes , Animais , Apoptose , Imunidade , Leucócitos , América do SulRESUMO
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.