Investigation of the Pathophysiology of the New Zealand Black-footed Abalone (Haliotis iris)

Grandiosa, Roffi
Alfaro, Andrea
Mérien, Fabrice
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Doctor of Philosophy
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Auckland University of Technology

This study was conducted to improve the knowledge regarding the immunological aspects of the New Zealand Blackfooted abalone (H. iris), to anticipate future occurrence of disease and to establish environmental-friendly therapeutic strategies in regards to the H. iris immune system. An initial study to identify the immunological aspects were by conducting characterization upon haemocytes using a combination of classical and novel (flow cytometry with Sysmex XN-1000 and Muse® Cell analyser) techniques. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The ability to successfully prepare and preserve haemocyte cells for microscopy and flow cytometry is critical for further investigation of the abalone immune system. Therefore, the study to apply different antiaggregants and handling protocols was conducted upon New Zealand black-footed abalone (H. iris) haemocytes. Results showed that Alsever’s solution was an effective antiaggregant, whereas K2EDTA Microtainer® tubes was similarly as effective as Alsever’s solution. However, the influence of different mixing techniques of K2EDTA Microtainer® tubes should be noticed. Observation of the immune capacity in juvenile New Zealand Black-footed abalone (H. iris) was assessed by conducting an experiment with probiotic enriched diet. Two groups of abalone were fed over a four-month period with different diets: one control and the other a probiotic diet enriched with Exiguobacterium JHEb1), 7x105 (Vibrio JH1) and 4.10x108 (Enterococcus JHLDc) CFU g-1 feed. Results showed that in comparison to control abalone (fed with the same diet but no probiotics), the probiotic fed abalone had a significantly higher (p < 0.05) total haemocyte count (1.9x106 cells), higher viable cell counts (90.77%), higher reactive oxygen species (ROS) positive haemocyte cells (12.03%) and a higher amount of non-apoptotic cells (87.96%). Further, foot tissue samples were obtained for metabolomics GC-MS analysis. Six free amino acids (lysine, proline, asparagine, glutamine and serine), two fatty acid (adrenic acid and palmitoleic acid), three organic compounds (lactic acid, succinic acid and oxalic acid) were detected in a higher amount in the foot muscle tissue of the probiotic-fed abalone. In addition, probiotic-fed animals showed improved growth and survival compared to control fed abalone.
The immune response of the probiotic-fed abalone to an intramuscular Vibrio splendidus challenge was also observed. Each abalone in this experiment were challenged by 109 cells mL−1 of pathogenic bacteria. Probiotic-fed abalone had a significantly higher survival rate post challenge compared to non-probiotic fed abalone. It was observed that the infected probiotic-fed abalone had higher THCs, slightly lower proportions of haemocytes undergoing early apoptosis, and lower percentages of ROS-producing haemocytes compared to infected control-fed abalone. These results indicate that the probiotic diet enhances immune capacity by stimulating haematopoietic processes, with a simultaneous low-level upregulation of ROS production, as a priming mechanism of the antibacterial defence system. In addition, metabolite profiles of muscle tissues generated via GC-MS provided suggestions of a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. This study suggests probiotics as an immunostimulant strategy since it potentially induce a protective effect against bacterial diseases leading to enhanced production and sustainability of the growing New Zealand abalone industry.

Abalone , Immunology , Probiotics , Haliotis , Iris , Metabolomics
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