Author: marina02

Development and evaluation of an oral vaccine for protection against red seabream iridovirus (RSIV) in barramundi (Lates calcarifer)

Red seabream iridovirus (RSIV) is an important fish viral pathogen that infects more than 30 species of cultured marine fish and has caused severe economic losses in barramundi (Lates calcarifer) aquaculture in Asia. Vaccination is an effective strategy to protect fish from viral infections. In this study, an inactivated oral vaccine with commercial adjuvant for preventing RSIV infection was developed. No histological changes were observed in the organs of barramundi immunized with the vaccine. In challenge tests, the cumulative mortality of barramundi immunized with the vaccine (40%) was lower than that of the control group (95%). Following vaccination, the mRNA expression levels of immune-related genes, including TNF-α, Mx, MHC-I, MHC-II, CD8, IgM, and IgT were significantly upregulated in the vaccine group compared to the control group. In the neutralization assay, the fish sera of the immunized barramundi exhibited a higher neutralizing antibody titer compared to the control group. These results suggest that the inactivated RSIV oral vaccine is safe for barramundi and could provide effective protection against RSIV infection.

1. CHANG, CHUN-KUEI, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, Presenter
2. WU, YU-CHI, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, Author

Development of a cross-protective bivalent glycovaccine against antimicrobial-resistant Streptococcus agalactiae and Streptococcus iniae for Finfish Aquaculture

Introduction

In low- and middle-income countries, aquaculture is a significant commercial commodity and a prospective source of food and livelihood. Streptococcus agalactiae and Streptococcus iniae, the major antimicrobial resistance (AMR) pathogens and causative agents for disease outbreaks inIndia resulting high mortality and economic loss. Moreover, the zoonotic potential of Streptococcus generates concerns about human health and the spread of AMR. Vaccines have been proven as a safe and targeted therapy against zoonotic multidrug-resistant Streptococcus to reduce the use of antibiotics. In the present study, we aimed to develop and validate the effectiveness of a bivalent cross-protective lectin-based vaccine against S. agalactiae and S. iniae comparing i.p, immersion and oral routes of administration.

Methodology

Isolated virulent strains of both pathogens will be grown in tryptic soy broth (TSB) brothand treated with 0.5% formalin. Lectin adjuvanted formalin killed vaccines will be used for immunization in fish via injection, immersion and followed by a booster vaccination on day 22. A virulent strain of S. agalactiae and S. iniae was injected to vaccinated fish and subsequent mortality was measured for 21 days. The fishes were sampled at fixed time intervals to perform ELISA to measure IgM antibody titers, histopathological examinations and immune gene expression using RT- PCR.

Results

Immune parameters, CD4, IgM, MHC-I, MHC-II, TCRβ, IL-1β, IL-6, and IL-8, were found to upregulate significantly (P < 0.05) in the spleen and head kidney of the vaccinated fish. Specific IgM antibody level was enhanced in immunized fish by ELISA. Aggregation of lymphocytes in gill and gut suggested the mucosal immune responses. Relative percentage survival was indicative of the protective efficacy of the bivalent vaccine. The vaccine was also found to be cross protective among the S. agalactiae 1a, 1b and III.

Conclusion

The study emphasizes a plausible approach to underpin the efficacy of the bivalent vaccine against the disease caused by S. agalactiae and S. iniae. The research indicates the cross-protection efficacy against both infections will be effective in limiting the spread of antimicrobial resistance.

Acknowledgments: For this study, the authors deeply acknowledge the funding supported by BactiVac, UK (BVNCP-5) and India-Taiwan collaboration program (2023/IN-TW/07).

1. ELUMALAI, PREETHAM, COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY, Presenter
2. Thompson, Kim D., Moredun Research Institute, Author
3. Desbois, Andrew P., Institute of Aquaculture, Author
4. Monaghan, Sean J., Institute of Aquaculture, Author
5. Pirarat, Nopadon, Chulalongkorn University, Author
6. Horton, Daniel, Institute of Aquaculture, Author
7. Subramani, Parasuraman Aiya, Thünen Institute of Fisheries Ecology, Author
8. Lakshmi, Sreeja, King Nandhivarman College of Arts and Science, Author

Single cell transcriptome sequencing of brown trout (Salmo trutta) immune cells – a baseline study

Profiling gene expression at the single-cell level offers unprecedented insights into the vertebrate immune system. While such studies in non-model organisms present unique challenges – such as evolutionary peculiarities – high-resolution data can yield valuable knowledge about ecologically and economically important species.

In this study, we used single-cell RNA sequencing (scRNAseq) to characterize the immune transcriptome of brown trout (Salmo trutta), a threatened indicator species commonly used to supplement endangered wild populations. We sampled multiple individuals from three distinct rearing backgrounds: wild-caught, farm-reared for one generation, and farm-reared for at least three generations.

Our analysis identified a comprehensive set of immune cell markers that account for these varied rearing histories, revealing a broadly conserved immune repertoire with lineage-specific differences in genes associated with neutrophils, macrophages, T cells, B cells, and other cell types. We also examined the evolutionary fate of gene duplicates resulting from the salmonid-specific whole-genome duplication event. Roughly half of these duplicates exhibited signs of functional divergence, such as differential expression across immune cell clusters.

Finally, by comparing gene expression at the cluster level across rearing backgrounds, we found that both short- and long-term exposure to farm environments induces transcriptional changes in key immune cells – including neutrophils, macrophages, B cells, and T cells – with some gene expression shifts shared across multiple lineages.

1. Schmidt-Posthaus, Heike, Institute for Fish and Wildlife Health, University of Bern, Presenter
2. Ord, James, Institute for Fish and Wildlife Health, University of Bern, Author
3. Saura Martinez, Helena, Institute for Fish and Wildlife Health, University of Bern, Author
4. Coxon, Quinn, Institute for Fish and Wildlife Health, University of Bern, Author
5. Talker, Stephanie, Institute of Virology and Immunology, University of Bern, Author
6. Adrian-Kalchhauser, Irene, Institute for Fish and Wildlife Health, University of Bern, Author

Modulation of rainbow trout (Oncorhynchus mykiss) immune response by fliC expression in Yersinia ruckeri

Introduction:

Enteric redmouth disease (ERM), caused by Yersinia ruckeri, presents a major challenge to salmonid aquaculture globally, resulting in considerable economic losses and compromising fish health. A key virulence factor of Y. ruckeri is its flagellum, which facilitates bacterial mo-tility, adhesion, and host invasion. The flagellin protein, encoded in part by the fliC gene, is integral to flagellar structure and function. While the involvement of flagella in bacterial path-ogenesis is well-documented, the specific immunomodulatory effects of fliC expression in rainbow trout (Oncorhynchus mykiss) remain insufficiently characterized. This study explores how variations in fliC expression among Y. ruckeri strains influence the immune responses of infected rainbow trout.

Methodology:

Rainbow trout were randomly distributed into three experimental groups: fliC-positive (fliC+), fliC-negative (fliC−), and a non-infected control. Each fish in the infected groups received an intraperitoneal injection of 2 × 10⁶ CFU of the Y. ruckeri strains, while control fish received phosphate-buffered saline (PBS). After 14 days, blood and tissue samples were collected for immunological assessments. Serum parameters, including lysozyme and ceruloplasmin activity, total protein, and immunoglobulin levels (γ-globulins), were quantified. Furthermore, spleen phagocyte respiratory burst and killing activity, as well as head kidney lymphocyte prolifera-tion (T and B cells), were evaluated.

Results:

Fish infected with the fliC− strain displayed significantly reduced lysozyme activity and γ-globulin levels relative to both the fliC+ and control groups. Respiratory burst activity in spleen phagocytes was markedly diminished in the fliC− group; however, these fish exhibited increased phagocytic killing activity. A significant decrease in T cell proliferation was also observed in the fliC− group, while B cell proliferation remained comparable across all groups.

Conclusions:

These results underscore the critical role of fliC expression in modulating the immune response of rainbow trout during Y. ruckeri infection. The data suggest that flagellin-mediated signaling contributes to both innate and adaptive immune responses, emphasizing the importance of flagellar components in the pathogenesis of enteric redmouth disease.

Funding:

This research was supported by project no. POIR.01.01.01-00-0906/18, “Highly efficient bac-teriophage screening using cytofluorimetric sorting in order to develop innovative products,” funded under Measure 1.1 “R&D projects of enterprises,” Sub-measure 1.1.1 “Industrial re-search and development work carried out by enterprises,” within the Smart Growth Opera-tional Program 2014–2020, co-financed by the European Regional Development Fund. Addi-tional support was provided by the National Inland Fisheries Research Institute in Olsztyn under the statutory research program No. Z-010: “Improving fish health protection methods in different rearing systems.”

1. SCHULZ, PATRYCJA, NATIONAL INLAND FISHERIES RESEARCH INSTITUTE, Presenter
2. PAJDAK-CZAUS, JOANNA, UNIVERSITY OF WARMIA AND MAZURY IN OLSZTYN, Presenter
3. POSPIECH, KAROLINA, PROTEON PHARMACEUTICALS S.A., Author
4. FORNAL, ELŻBIETA, PROTEON PHARMACEUTICALS S.A., Author
5. KOBIERA, AMANDA, PROTEON PHARMACEUTICALS S.A., Author
6. MATCZAK, JUSTYNA, PROTEON PHARMACEUTICALS S.A., Author
7. FOKSIŃSKI, PAWEŁ, UNIVERSITY OF WARMIA AND MAZURY IN OLSZTYN, Author
8. SIWICKI, ANDRZEJ, NATIONAL INLAND FISHERIES RESEARCH INSTITUTE, Author

Evaluation of the potential effects of sodium fluoride, a suspected endocrine disrupting substance, on the immune system of rainbow trout, Oncorhynchus mykiss – SUSPECT

Introduction:

The sanitary authorities have a particular interest in endocrine disrupting compounds (EDCs) because of their increasing use and their proven and/or suspected harmful effects on humans and the environment. Historically, the first effects of EDCs were demonstrated on the metabolism of sex steroid hormones. More recently, it has been shown that they can disrupt the balance of thyroid hormones (TH) in vertebrates. These THs play a major role in several physiological and metabolic processes and act as modulators of immune responses (IR), with impacts on several key mechanisms such as phagocytosis or the synthesis/release of cytokines, as demonstrated in hypo or hyperthyroid conditions. IR can also be influenced by the different microbiota associated with the host. In teleosts, any disturbance inducing dysbiosis can affect the fitness of fish, particularly by increasing their susceptibility to pathogens. In an open aquatic environment carrying many chemical and biological pollutants, immunity represents a key defense element whose effectiveness can therefore potentially be modulated by the thyroid hormone system (THS) and these different microbial populations. Our project aims to generate scientific data on the impact of a suspected EDC, sodium fluoride (NaF), on a model freshwater fish, rainbow trout (RT), by targeting the effects on and the interactions between the THS, the gill and intestinal microbiota and the IR.

Methodology:

A comparative study between NaF and a proven endocrine disruptor, tebuconazole (TBZ), was carried out during the first months of the trout’s life, using a multiparametric approach. Following 8 months of chronic exposure to different concentrations of these EDCs, fish were infected with infectious hematopoietic necrosis virus (IHNV) to assess their potential for resistance. During chemical exposure and then infection, fish mortality and growth were monitored regularly, and various organ and blood samples were taken for future analysis. Classic immune parameters were measured by cellular hematology methods (cell counts, leucocyte differentiation) and flow cytometry (phagocytosis, necrosis/apoptosis), humoral biomarkers by spectrometry (lysozyme, complement activity, antibody secretion) and transcriptomic approaches by qPCR in spleen, i.e. gene expressions of the complement system and pro-inflammatory (IL1β, TNFα, IFN).

Results:

The assays are ongoing and results obtained on the IR of RT exposed to these suspected EDCs will be presented and discussed.

Keywords: Immune system, Rainbow trout, Complement, Viral challenge, Antibodies

1. LANNUZEL, Guillaume, ANSES LABORATOIRE DE PLOUFRAGAN PLOUZANÉ NIORT UNITÉ VIMEP, Author
2. DANION, Morgane, ANSES LABORATOIRE DE PLOUFRAGAN PLOUZANÉ NIORT UNITÉ VIMEP, Author
3. LOUBOUTIN, Lenaig, ANSES LABORATOIRE DE PLOUFRAGAN PLOUZANÉ NIORT UNITÉ VIMEP, Presenter
4. MORIN, Thierry, ANSES LABORATOIRE DE PLOUFRAGAN PLOUZANÉ NIORT UNITÉ VIMEP, Author
5. BRAUNBECK, Thomas, Aquatic Ecology and Toxicology Section, Center for Organismal Studies, University of Heidelberg, Germany, Author
6. BELLEC, Laure, Université de Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, Pessac, France, Author
7. PANNETIER, Pauline, ANSES LABORATOIRE DE PLOUFRAGAN PLOUZANÉ NIORT UNITÉ VIMEP, Author

Single-cell sequencing of immune cells provides insights into the early immune response of brown trout (Salmo trutta) to Tetracapsuloides bryosalmonae infection

Understanding how environmental factors shape immune responses is essential to protecting vulnerable fish populations in a changing climate. Brown trout (Salmo trutta) are both an ecologically important species as apex predators in their native habitats and an economically important species for recreational anglers and commercial fisheries. Wild trout populations are under significant threat from Proliferative Kidney Disease (PKD), caused by the myxozoan parasite Tetracapsuloides bryosalmonae. To combat population decline, restocking programs are implemented involving the large-scale release of captive-raised fish. However, it is unclear, whether this measure is supportive to the population, especially in presence of PKD, as captive-raised fish may be more susceptible to disease.

Mechanisms underlying differences in susceptibility to PKD between wild and captive-reared trout remain poorly understood. Single-cell RNA sequencing (scRNA-seq) offers us a unique opportunity to profile the trout immune response at cellular resolution. We conducted a controlled infection experiment with 18 individuals from different rearing backgrounds: wild, farmed, and mixed (farm-born of wild parentage). scRNA-seq of kidney tissue revealed 113 genes differentially expressed between infected and control groups across immune cell types. Notably, the most prominent transcriptional changes were observed in neutrophils, B cells, and CD4+ T cells.

Our findings provide the first high-resolution map of trout immune cell responses to T. bryosalmonae. They also reveal the fate of duplicate genes unique to the salmonid lineage, of which approximately half showed a different expression pattern from their paralogs. These insights advance our understanding of the trout response to PKD and can help inform future decisions on the best methods of combating the disease. More broadly, they contribute to the growing understanding of the poorly characterized salmonid immune system.

1. Coxon, Quinn, University of Bern, VetSuisse, Presenter
2. Saura Martinezez, Helena, University of Bern, VetSuisse, Author
3. Ord, James, University of Helsinki, Author
4. Adrian-Kalchhauser, Irene, University of Bern, VetSuisse, Author
5. Schmidt-Posthaus, Heike, University of Bern, VetSuisse, Author

Comparison of Initial Immune Responses among Six Salmonid Species Inoculated with PolyI:C

Introduction: During infection, various responses are induced to eliminate the pathogen. In this study, we aimed to identify antiviral responses commonly important across salmonid species. For comparison across salmonid species, we selected six salmonid species in four different genera and examined gene expression in the spleens of the salmonids following inoculation with polyI:C, which mimics viral infection. We identified genes up-/down-regulated in response to polyI:C stimulation and compared the initial immune responses among species based on the function of the differentially expressed genes (DEGs).

Methodology: Six salmonid species (rainbow trout, Oncorhynchus mykiss; masu salmon, O. masou; coho salmon, O. kisutch; Japanese char, Salvelinus leucomaenis; brown trout, Salmo trutta; whitefish, Coregonus maraena) were inoculated intraperitoneally with polyI:C (25 µg/g body weight). Total RNA was extracted from the spleen at 6 and 12 h post-inoculation and subjected to RNA-seq. Gene expression levels in the polyI:C-stimulated group were compared with those in the PBS-injected control group. The expression patterns of the orthologs common to six species were analyzed using principal component analysis (PCA) of the normalized read counts. Enrichment analysis was performed on the DEGs to compare the induced responses, GO term and KEGG pathway. Subsequently, we compared the expression patterns of the genes involved in those responses in at least one species and their orthologs in the other species.

Results: In all the salmonid species examined, gene ontologies “immune response” and “defense response to virus” and KEGG pathways related to pattern recognition systems were overrepresented following polyI:C inoculation. The Salmoninae and Coregoninae subfamilies were separated from each other, whereas there were no differences among the genera of Oncorhynchus, Salvelinus, and Salmo within the Salmoninae subfamily in the PCA plot. The DEGs common to the six species included type I interferons (IFNs) and many orthologs of mammalian IFN-regulated genes (IRGs). The subfamilies Salmoninae and Coregoninae showed different patterns in the expression of genes encoding receptors and inflammatory-related molecules belonging to the pattern recognition systems.

Conclusion: Our study using polyI:C stimulation suggests that the expression of type I IFNs and IRGs is commonly induced in the six salmonid species during viral infection. On the other hand, the Salmoninae and Coregoninae subfamilies are speculated to differ in expression patterns of genes associated with viral infection, particularly in expression patterns of pattern recognition-related genes.

Funding: This study was funded by the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, Fisheries of Japan.

1. MORIMOTO, Natsuki, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Presenter
2. UMEDA, Kousuke, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Author
3. TAKANO, Tomokazu, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Author
4. MATSUYAMA, Tomomasa, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Author

Genomic analysis and rtp3 gene expression in gilthead sea bream (Sparus aurata) following NNV infections

Introduction

Receptor-transporting protein 3, rtp3, is an interferon-stimulated gene with potential antiviral functions in fish. However, the genomic organization of fish rtp3 genes and their role in the antiviral defence remain poorly characterized. Nervous necrosis virus (NNV), a member of the Betanodavirus genus, is the causative agent of viral nervous necrosis. Four NNV genotypes have been identified, as well as reassortant strains, such as RGNNV/SJNNV. Notably, only SJNNV and RGNNV/SJNNV viruses cause mortality in gilthead sea bream (Sparus aurata).

Aim

Characterization of the genomic organization of gilthead sea bream rtp3 genes and analysis of their transcription in response to NNV infections. This is a first step for future research aimed to develop measures to prevent NNV infections in this fish species

Material and Methods

Sea bream rtp3 sequences were identified by BLAST analysis, using the sea bream reference genome (GenBank: GCA_900880675.2). Untranslated regions, introns, and amino acid sequences were predicted using bioinformatics tools. Phylogenetic analysis was performed through the protein sequence alignment. For in vivo analysis, fish were injected with: (i) poly I:C; (ii) RGNNV; or (iii) SaNNV, RGNNV/SJNNV. Basal rtp3 transcription was established across multiple organs in untreated fish. Poly I:C-induced transcription was studied in head kidney and brain and NNV-induced transcription was studied in brain at different times post infection.

Results

This study has identified three sea bream rtp3 genes (sa-rtp3-X1, sa-rtp3-X2, sa-rtp3-X3) exhibiting distinct structure: sa-rtp3-X1 and sa-rtp3-X3 share a two-exon structure; whereas sa-rtp3-X2 contains three exons. The three promoters share conserved immune-related regulatory elements, including ISRE-like sequences and GAAA/TTTC repeats, supported by high sequence conservation with other teleost rtp3 genes. Basal expression profiling showed that sa-rtp3-X1 and sa-rtp3-X3 were widely transcribed in head kidney, intestine and spleen, whereas sa-rtp3-X2 transcription was predominantly restricted to head kidney. All sa-rtp3 genes responded to poly I:C, displaying distinct induction kinetics in head kidney and brain. In viral challenge experiments, both viruses induced sa-rtp3-X1 and sa-rtp3-X3, whereas sa-rtp3-X2 remained mostly unresponsive.

Conclusions

These results demonstrate the constitutive and inducible transcription of sea bream rtp3 genes, supporting their involvement in antiviral immunity. The differential regulation and structure of these genes suggest functional specialization concerning the sea bream defense against NNV, providing a molecular framework for future studies in this high-value aquaculture species.

Funding: Agencia Estatal de Investigación. Ref. PID2020-115954RB-I00

1. Alvarez-Torres, Daniel, University of Malaga, Author
2. Moreno, Patricia, University of Malaga, Author
3. Juan, Gemez-Mata, University of Malaga, Author
4. Garcia-Rosado, Esther, University of Malaga, Author
5. Bejar, Julia, University of Malaga, Author
6. Alonso, M. Camen, University of Malaga, Presenter

Modified flagellin retains immunostimulatory activity, bridging innate and adaptive immunity in trout (Oncorhynchus mykiss)

The aquaculture industry demands efficient adjuvants to enhance fish vaccines against bacterial and viral pathogens. Flagellin has been suggested as a potential immunostimulant and vaccine adjuvant due to its ability to induce Th1 and Th2 responses. Moreover, flagellin acts as an antigen, inducing neutralizing antibodies when used continuously. To avoid this problem, various modifications, such as deletions, have been carried out while preserving the interaction with TLR5. However, the adjuvant capacity of flagellins with the deleted hypervariable region in fish remains to be elucidated. Our previous results have shown that both full-length flagellin and hypervariable domain (HPVD)-deleted flagellin from Marinobacter algicola can significantly increase the transcription of IL1beta, IL6, IL8, IFNg, and TNFa in RTS-11 and gut culture cells.

In the present study, we investigated the immunostimulant effects of full-length and HPVD-deleted flagellins in trout (Oncorhynchus mykiss) fry. Both flagellins were expressed in E. coli and further purified by His-tag affinity chromatography. Trout were injected intraperitoneally with 5 micrograms of each flagellin, PBS, and control protein to assess LPS during protein purification. Spleens were harvested 6 and 12 hours after injection (hpi), RNA was extracted, and cDNA was synthesized. Gene expression of some genes involved in immune response was further analyzed and quantified by the 2^-deltaCT method. It was found that both flagellins induced the expression of IL1beta, IL6, IL8 and IFNg in the spleen at a higher rate at 6 hpi than at 12 hpi compared to the control group. The expression of genes such as CCR7 and IL17A1 is not observed at 6 hpi, but rather, these genes demonstrate significant expression at 12 hpi. Other genes, including TNFa, CD80/86, MHC Class I, and IL17C, did not increase their expression at either 6 hpi or 12 hpi, while CD80/86 decreased its expression at 12 hpi from its normal expression at 6 hpi. Both flagellins triggered the expression of some immune-related genes from innate and adaptive responses. In this regard, CCR7 plays an important role in the response of dendritic cells, their maturation, and interaction with T lymphocytes for the immune response.

The use of HPVD-deleted flagellin confers functional advantages over full-length flagellin in vivo, as it is effective in inducing an immunostimulatory response in trout, but induces fewer antibodies against itself. Therefore, the use of deleted flagellin as a vaccine adjuvant in fish is promising.

1. LÓPEZ, MARCELO, Department of Biotechnology. INIA-CSIC. 28040-Madrid, Spain, Presenter
2. GARCÍA, JOSÉ, Animal health faculty of veterinary. UCM. 28040-Madrid, Spain, Author
3. GOMEZ-CASADO, EDUARDO, Department of Biotechnology. INIA-CSIC. 28040-Madrid, Spain, Author

Protocol optimization for leucocytes isolation from the blood of Atlantic salmon

Isolation of peripheral blood leukocytes (PBL) is an important tool for studying immune responses and understanding the infection dynamics. Isolation of macrophages from Atlantic salmon head kidney has been described in several studies while fewer studies have been used the isolation of PBLs.

Here we report optimization of the protocols used for isolation of PBLs from Atlantic salmon (Salmo salar). Blood samples were collected from different individuals using heparinized blood vacutainer. The blood was then diluted with either PBS or 0.9% NaCl in 1/3 and 2/3 ratio. After dilution, the blood was layered slowly on top of either 37% or 54% Percoll or Lymphocyte Separation Medium (LSM) containing poly-sucrose and sodium diatrizoate adjusted to a density of 1.0770 +/- 0.0010 g/ml. The cells were then centrifuged at 4˚C and 400g for 30 min. The centrifugation time has been increased if complete separation was not obtained. The cell layer was then carefully collected using Pasteur pipette and placed in a new collection tube. Cells were then washed twice in PBS or 0.9 NaCl using repeated rounds of centrifugation. The number of cells and viability were assayed by the trypan exclusion assay using an automated cell counter. In addition, smears were prepared from pelleted cells to determine the isolated cell populations by differential staining techniques using Giemsa stain.

The findings and results of the study will be presented and discussed.

1. Radunovic, Sandra, NORWEGIAN UNIVERSITY OF LIFE SCIENCES, Presenter
2. Gamil, Amr, Norwegian University of Life Sciences, Author
3. Evensen, Øystein, Norwegian University of Life Sciences, Author