Elsevier

Fish & Shellfish Immunology

Volume 88, May 2019, Pages 179-188
Fish & Shellfish Immunology

Full length article
Characterisation of Chinook salmon (Oncorhynchus tshawytscha) blood and validation of flow cytometry cell count and viability assay kit

https://doi.org/10.1016/j.fsi.2019.02.059Get rights and content

Highlights

  • Salmon smolts peripheral blood total cell count was estimated at 2.3 × 106 μL−1.

  • Differential cell counts revealed five cell types dominated by 97.18% erythrocytes.

  • Fish PBMCs successfully isolated from a micro blood volume (<300 μL).

  • Isolated PBMCs were majorly composed of lymphocytes and monocytes.

  • Muse® Cell Analyser delivered accurate and precise results as the haemocytometer.

Abstract

New Zealand Chinook salmon (Oncorhynchus tshawytscha) industry has great potential for growth and expansion. While production is relatively free of health problems, there is limited literature on haematology, and immunological tools to safeguard against possible future health threats. The current study aim was to characterise New Zealand farmed O. tshawytscha peripheral blood cellular composition, develop a micro-volume method to isolate peripheral blood mononuclear cells (PBMCs) and validate a microcapillary flow cytometry assay kit for PBMC cell count and viability assessment. We used light microscopy to characterise peripheral blood and PBMC cellular composition in combination with a flow cytometer Sysmex XT 2000i Haematology Analyser. ImageJ version 1.52 was used for cell size characterisation of freshly stained blood. The stability of PBMCs stained with the Muse® Cell Count and Viability Assay Kit and the Trypan blue assay stains were studied at 4 °C and 21 °C for 60 min; while the Muse® Cell Count and Viability Assay Kit was validated against the Trypan blue assay haemocytometer chamber to assess PBMC count and viability. Findings showed that O. tshawytscha smolt yearlings had total blood cell counts in the range of 1.9–2.7 × 106 μL−1. Differential cell counts revealed five cell types, comprising 97.18% erythrocytes, 2.03% lymphocytes, 0.67% thrombocytes, 0.09% monocytes, and unquantifiable neutrophils. Using micro-volumes of blood and Lymphoprep™, we successfully isolated fish PBMCs. Significantly, stained PBMCs remained stable for up to 45 min at 4 °C and 21 °C; while validation of the Muse® protocol showed that this microfluidic instrument delivered more accurate and precise viability results than the haemocytometer. The Muse® protocol is rapid, easy to use, has quick calibration steps, and is suitable for field use to facilitate onsite sample processing. These findings pave the way for future assessments of fish health and in vitro immunological studies in O. tshawytscha.

Introduction

Immunity is an integrated physiological and barrier system that protects animals from threats posed by pathogens [1,2]. The vertebrate immune machinery consists of the innate and adaptive immune systems; and teleosts are the only vertebrate ancestral group that possess both systems [3]. The innate immune response consists of physical barriers (e.g. the skin, scales, gut mucosa and gill epithelia), humoral and cellular components. This system acts as the first line of defence against pathogens. The humoral component includes complement proteins, lysozymes, proteases, esterases, antimicrobial peptides (AMPs) and immunoglobulins, like IgM, IgD and IgT or IgZ [4,5]. The cellular immune constituents include leucocytes, such as T and B lymphocytes, neutrophils, eosinophils, basophils, thrombocytes, cytotoxic cells (natural killer cells), mast cells (MCs), dendritic cells (DCs), macrophages and their precursor monocytes [6,7]. Neutrophils, eosinophils and basophils have a granulated cytoplasm, thus called granulocytes [8]. Macrophages and neutrophils are professional phagocytes [9,10] with a high phagocytic ability and capacity [10]. These cells produce antimicrobial nitrogen and oxygen products, cytokines, and ingest a wide range of particle sizes [11].

When the physical barriers fail to prevent pathogen attacks and entry, other innate immune components become involved through pattern recognition receptors (PRRs) on cell surfaces, which recognise common conserved pathogen-associated molecular patterns (PAMPs) not expressed in multicellular organisms [6,12]. These PAMPs include viral RNA and bacterial DNA, fungal β1,3-glucans, bacterial cell wall peptidoglycans, polysaccharides and lipopolysaccharides (LPS) [12]. The recognition of PAMPs initiates cellular responses designed to kill and eliminate microbial pathogens [13] via phagocytosis and exocytosis [14]. Phagocytosis initiates release of cytokines, followed by antigen presentation through the major histocompatibility complex (MHC), resulting in the development of adaptive immunity [15]. Overall, the adaptive immune component relies on the humoral (antibody) and cellular responses, and is characterised by specific antigen recognition, which invokes a quick and strong secondary pathogen-specific response [15].

In aquaculture, disease outbreaks caused by viruses, bacteria, and parasites, routinely lead to heavy industry losses [reviewed in Ref. [16]]. Amidst these challenges, aquaculture has gained significant progress, and is presently recognised as one of the fastest growing food production sectors worldwide [17]. To keep up with demand, aquaculture farms aim to produce high quantity, of healthy and fast-growing fish, under optimal husbandry and management practices. Combined with other diagnostic tools, fish haematology can reveal important information on fish physiology and health, with high application potential to investigate and monitor stress responses, diagnose diseases and identify nutritional problems [reviewed in Ref. [16]]. For instance, haematology combined with humoral, cellular immune parameters and gene expression have been employed in several fish species to demonstrate immunomodulatory effects of: dietary supplements [18,19]; immunostimulants and pathogen challenge [[20], [21], [22], [23]]; temperature perturbations [[24], [25], [26]]; and hypoxia [27]. Furthermore, salinity stress [28]; crowding stress [29]; heavy metals [30,31]; hormonal components [32]; and melatonin [33] have been shown to induce dramatic changes in leucocyte counts.

However, most fish immunology studies are conducted in vivo, which can be time consuming, costly, and often involve fish euthanasia. Thus, there is a need for alternative models that are less invasive, can be performed under controlled in vitro environments, with nominal bio-fluid or tissue requirements, and which are less time consuming and more affordable. To perform in vitro immunological studies, fish immune cells are sourced from the thymus, kidney, spleen, or peripheral blood [34]. These cells can be isolated from lymphoid tissues and blood by either hypotonic lysis or density gradient centrifugation [35,36]. Indeed, isolated peripheral blood mononuclear cells (PBMCs) composed of suspended lymphocytes and adherent monocytes have been previously used to investigate immunomodulatory effects of: dietary supplements and disease resistance [18,37]; pathogen challenge [22,38], and vaccine efficacy [23,39,40], among others. Thus, fish PBMCs present an important opportunity to model several biological variables via integrated techniques, including microscopy, flow cytometry, RT-qPCR, spectrophotometry, western blotting, among others.

Specifically, flow cytometry has been used to characterise fish PBMCs in rainbow trout (Oncorhynchus mykiss) [35], sea bass (Dicentrarchus labrax) [41], striped catfish (Pangasius hypothalamus), and European eel (Anguilla anguilla) [36]. Flow cytometry has also been used to assess PBMCs for: production of reactive oxygen species (ROS) [22,42]; antibody detection [43,44]; phagocytosis [26,42,45,46] and natural cytotoxicity [47]. Consequently, flow cytometry tools such as the Muse® Cell Analyser, offer accurate and fast quantitative assessment of single cells compared to traditional methods. Fortunately, the Muse® Cell Analyser has been successfully used in our laboratory to characterise haemocytes in black-footed abalone (Haliotis iris) [48,49]; to study immune responses of shellfish to pathogen infections [50]; T. V. [51]; T. V [[50], [51], [52]]. and stressors [53]; T. [[53], [54]]; and to assess the immune status of spiny lobster (Jasus edwardsii) with tail fan necrosis [55].

While the Muse® Cell Analyser has been shown to work well with shellfish haemolymph, the platform has not yet been used on fish blood, which first requires isolation of PBMCs to exclude nucleated erythrocytes. Fish PBMCs have previously been isolated for immunological investigations in Atlantic salmon (Salmo salar) [43,45], and in Chinook salmon (Oncorhynchus tshawytscha) in Oregon state USA [56], but this information is still lacking for cultured O. tshawytscha in New Zealand. This presents an opportunity to incorporate the Muse® Cell Analyser as a fish health assessment tool for the aquaculture industry and other research-based applications.

The New Zealand aquaculture sector was valued at over US$ 800 million in 2016 of which O. tshawytscha contributed 22% and 12% by value and production volume respectively [57]. The species is the second most important aquaculture product after the Greenshell™ mussel (Perna canaliculus), making New Zealand, a global leader in production and supply. Despite the economic importance of O. tshawytscha, there is a lack of literature on haematological and immunological aspects of this species. Continued growth of the salmon industry will necessitate species-specific immunological information and development of tools to monitor and assess fish health. Thus, the present study characterised the cellular composition of O. tshawytscha peripheral blood, and developed a micro-volume method to isolate fish PBMCs. We also characterised isolated PBMCs and used them to validate a commercial cell count and viability assay kit using the Muse® Cell Analyser.

Section snippets

Fish samples

Nineteen yearling smolts (weight = 317.2–544.5 g; total length = 23.7–28.0 cm) were obtained from the Nelson Marlborough Institute of Technology (NMIT) aquaculture facility (Glenduan, Nelson, New Zealand). Fish had been maintained on a saltwater recirculating system (temp = 14.26 ± 0.35 °C; DO = 7.30 ± 0.11 mgL−1; pH = 8.12 ± 0.21; NH4 = 0.14 ± 0.13 mgL−1; NO2 = 0.28 ± 1.12 mgL−1), and they were fed to satiation daily with EWOS Microboost 2.2 mm commercial diet (Cargill) with 50% crude

Peripheral blood cellular characterisation

Five types of cells in Giemsa-stained peripheral blood were identified, comprising erythrocytes, lymphocytes, thrombocytes, monocytes and neutrophils. Neutrophils were observed, but not sufficiently quantifiable. Generally, erythrocytes dominated cell composition (Fig. 1). The high dominance of erythrocytes was also confirmed via differential cell counts where total leucocytes accounted for less than 3%, and majority were lymphocytes (Table 1).

Erythrocytes were the largest cells with ellipsoid,

Discussion

In this study, we characterised peripheral blood cells in O. tshawytscha yearling smolts using light microscopy and flow cytometry, we successfully established a micro blood volume (<300 μL) density gradient centrifugation method to isolate and purify PBMCs, and validated an efficient assay kit to assess PBMC count and viability using a portable flow cytometry platform, the Muse® Cell Analyser. Differential cell count revealed five types of peripheral blood cells, consisting of erythrocytes,

Acknowledgements

The authors would like to thank Leanne D Jones of the Nelson Marlborough Institute of Technology (NMIT) for her generous support in maintaining the study animals during laboratory sessions at the Glenduan Aquaculture Park in Nelson, New Zealand. We are also thankful to the technical support at the Auckland University of Technology, particularly Meena Patel, Kathryn Hattersley at NMIT medical laboratory and the Cawthron Institute during the course of this study. This work was greatly improved by

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