Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

C Collins; M Hall; D Bruno; J Sokolowska; L Duncan; R Yuecel; U McCarthy; M J Fordyce; C C Pert; R McIntosh; Z MacKay

doi:10.1111/jfd.12517

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

C Collins, M Hall, D Bruno, J Sokolowska, L Duncan, R Yuecel, U McCarthy, M J Fordyce, C C Pert, R McIntosh, Z MacKay

Applied Medicine

MARINE SCOTLAND SCIENCE

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21 Citations (Scopus)

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Abstract

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

Original language	English
Pages (from-to)	351–365
Number of pages	15
Journal	Journal of Fish Diseases
Volume	40
Issue number	3
Early online date	15 Aug 2016
DOIs	https://doi.org/10.1111/jfd.12517
Publication status	Published - 1 Mar 2017

Bibliographical note

The authors would like to thank Katherine Lester, Louise Feehan and Ottavia Benedicenti, Ben Williamson, Mark Patterson, Patricia Noguera and Bertrand Collet for help with aquarium challenge and sampling. The authors would also like to recognize the support of Marine Harvest (Scotland) Ltd in providing material for generation of initial Paramoeba perurans in vitro cultures. This work was funded by Scottish Government grant AQ0080.

Keywords

Paramoeba
perurans
flow cytometry
clonal
culture
virulence

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Accepted Manuscript
This is the peer reviewed version of the following article:Collins, C., Hall, M., Bruno, D., Sokolowska, J., Duncan, L., Yuecel, R., McCarthy, U., Fordyce, M. J., Pert, C. C., McIntosh, R. and MacKay, Z. (2017), Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence. J Fish Dis, 40: 351–365, which has been published in final form at DOI: 10.1111/jfd.12517. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Accepted author manuscript, 87.1 KBLicence: Unspecified

BD Influx Cell Sorter
Holme, A. (Manager)
Iain Fraser Cytometry Centre
Research Facilities: Equipment
BD LSR Fortessa
Holme, A. (Manager)
Iain Fraser Cytometry Centre
Research Facilities: Equipment
Iain Fraser Cytometry Centre
Holme, A. (Manager), Duncan, L. (Senior Application Scientist), Laird, A. (Technician) & Burgoyne, K. (Technician)
Institute of Medical Sciences
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Cite this

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title = "Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence",

abstract = "Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.",

keywords = "Paramoeba, perurans, flow cytometry, clonal, culture, virulence",

author = "C Collins and M Hall and D Bruno and J Sokolowska and L Duncan and R Yuecel and U McCarthy and Fordyce, {M J} and Pert, {C C} and R McIntosh and Z MacKay",

note = "The authors would like to thank Katherine Lester, Louise Feehan and Ottavia Benedicenti, Ben Williamson, Mark Patterson, Patricia Noguera and Bertrand Collet for help with aquarium challenge and sampling. The authors would also like to recognize the support of Marine Harvest (Scotland) Ltd in providing material for generation of initial Paramoeba perurans in vitro cultures. This work was funded by Scottish Government grant AQ0080.",

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AU - Hall, M

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AU - McCarthy, U

AU - Fordyce, M J

AU - Pert, C C

AU - McIntosh, R

AU - MacKay, Z

N1 - The authors would like to thank Katherine Lester, Louise Feehan and Ottavia Benedicenti, Ben Williamson, Mark Patterson, Patricia Noguera and Bertrand Collet for help with aquarium challenge and sampling. The authors would also like to recognize the support of Marine Harvest (Scotland) Ltd in providing material for generation of initial Paramoeba perurans in vitro cultures. This work was funded by Scottish Government grant AQ0080.

PY - 2017/3/1

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N2 - Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

AB - Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

KW - Paramoeba

KW - perurans

KW - flow cytometry

KW - clonal

KW - culture

KW - virulence

U2 - 10.1111/jfd.12517

DO - 10.1111/jfd.12517

M3 - Article

C2 - 27524425

SN - 0140-7775

VL - 40

SP - 351

EP - 365

JO - Journal of Fish Diseases

JF - Journal of Fish Diseases

IS - 3

ER -

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Abstract

Bibliographical note

Keywords

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Equipment

BD Influx Cell Sorter

BD LSR Fortessa

Iain Fraser Cytometry Centre

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