This article was downloaded by: [Van Pelt and Opie Library] On: 22 October 2014, At: 11:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Development and Characterization of a Largemouth Bass Cell Line a
a
a
b
Rodman G. Getchell , Geoffrey H. Groocock , Emily R. Cornwell , Vanessa L. Schumacher , a
a
c
a
Lindsay I. Glasner , Barry J. Baker , Stephen A. Frattini , Gregory A. Wooster & Paul R. a
Bowser a
Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA b
Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
c
The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030, USA Published online: 21 Aug 2014.
To cite this article: Rodman G. Getchell, Geoffrey H. Groocock, Emily R. Cornwell, Vanessa L. Schumacher, Lindsay I. Glasner, Barry J. Baker, Stephen A. Frattini, Gregory A. Wooster & Paul R. Bowser (2014) Development and Characterization of a Largemouth Bass Cell Line, Journal of Aquatic Animal Health, 26:3, 194-201, DOI: 10.1080/08997659.2014.922517 To link to this article: http://dx.doi.org/10.1080/08997659.2014.922517
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:194–201, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.922517
ARTICLE
Development and Characterization of a Largemouth Bass Cell Line Rodman G. Getchell,* Geoffrey H. Groocock, and Emily R. Cornwell Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Vanessa L. Schumacher Downloaded by [Van Pelt and Opie Library] at 11:25 22 October 2014
Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
Lindsay I. Glasner and Barry J. Baker Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Stephen A. Frattini The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030, USA
Gregory A. Wooster and Paul R. Bowser Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22◦ C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week.
Cell lines provide an important diagnostic tool even though the availability of molecular assays is increasing. Close to 300 cell lines from finfish have been established since the first gonadal cell line was developed from Rainbow Trout Oncorhynchus mykiss (RTG-2; Wolf and Quimby 1962; Fryer and Lannan 1994; Lakra et al. 2011). During the last 20 years, 60% of the new cell lines have come from Asian investigators, where *Corresponding author: [email protected] Received February 18, 2014; accepted April 16, 2014
194
more than 80% of world aquaculture products originate (Lakra et al. 2011). As a result of its popularity as a game fish, Largemouth Bass Micropterus salmoides may now be the most widely introduced fish in North America (USDA 2006). Largemouth Bass are cultured at private, state, and federal facilities for recreational fishing and stocking programs. According to the 2005 Census of
Downloaded by [Van Pelt and Opie Library] at 11:25 22 October 2014
NEW LARGEMOUTH BASS CELL LINE
Aquaculture (USDA 2006), 192 U.S. fish farms reared Largemouth Bass, posting total sales of US$10.6 million that year. Largemouth Bass were introduced in China in 1983 (Deng et al. 2011) and have become widely cultured with annual production levels over 100,000 tons (Bai et al. 2008). Prior to 2011, the only significant viral pathogen of Largemouth Bass was the iridovirus, Largemouth Bass virus (LMBV), which had been isolated from Largemouth Bass in the eastern USA since 1991 (Plumb et al. 1996; Grizzle et al. 2002). But recently, three new viruses have been isolated from cultured Largemouth Bass in China, a ranavirus (Deng et al. 2011), a megalocytivirus (Ma et al. 2011), and a rhabdovirus (Ma et al. 2013). In Italy, a betanodavirus has been isolated from cultured Largemouth Bass (Bovo et al. 2011). The emergence of viral pathogens remains a significant constraint for aquaculture production and for the sustainability of biodiversity in the natural environment (Sahoo and Goodwin 2012). As viruses cannot reproduce on their own, the development of new fish cell lines aids in the detection and characterization of these emerging pathogens. Largemouth Bass virus has been isolated from 16 species of fish in the USA (Iwanowicz et al. 2013) and will replicate in several cell lines, and optimal cell culture methods for the detection of this virus have been determined (Piaskoski et al. 1999; McClenahan et al. 2005). Five cell lines previously tested for their LMBV susceptibility are: Bluegill fry (BF-2; ATCC certified cell line [CCL]-91), Fathead Minnow (ATCC CCL42), epithelioma papulosum cyprini (ATCC cell repository line [CRL]-2872), Channel Catfish ovary (ATCC CRL-2772), and Chinook Salmon embryo (CHSE-214; ATCC CRL-1681). All five of these lines have similar susceptibility to LMBV infection (McClenahan et al. 2005). The objective of this study was to establish and characterize a new cell line from the gonad of Largemouth Bass (LMBG) and evaluate the replication of LMBV in this cell line versus BF-2 cells.
METHODS Fish.—Largemouth Bass of various sizes were captured in June 2004 from Oneida Lake, New York, by electrofishing and transported to the Cornell University Aquatic Animal Health Program. Twenty Largemouth Bass were euthanized and the gonads were removed for processing and culture. This work was conducted under an animal protocol approved by the Institutional Animal Care and Use Committee of Cornell University. Cell line initiation.—Gonads from each of 20 adult Largemouth Bass were collected aseptically and processed separately. Following a procedure Bowser and Plumb (1980) developed to create an ovary cell line from Channel Catfish Ictalurus punctatus, Largemouth Bass tissues were minced and suspended in 20 mL of Hank’s balanced salt solution (HBSS), and the suspension was centrifuged at 1,500 × g for 5 min. The HBSS was then decanted and tissues were resuspended in 5 mL of 0.25% trypsin. The tissue suspension was immediately transferred to a diges-
195
tion flask and an additional 15 mL of 0.25% trypsin added. Cells were later harvested in aliquots after 8 h of digestion with continuous mixing. Harvested cells were washed and resuspended in phosphate-buffered saline (PBS) three times. Finally, cells were suspended in Hank’s minimal essential media (HMEM) with 20% fetal bovine serum (FBS) and 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine (PSG), and then aliquoted into primary 25-cm2 culture flasks. Flasks were incubated at room temperature, at approximately 22◦ C (hereafter 22◦ C). The cell lines were weaned onto HMEM with 10% FBS by passage 20. The flasks containing cells that adhered and grew were propagated. None of the flasks with testicular cells remained viable from the few males that were processed. For the initial 4–5 years, the LMBG cells were very slow growing. Complete monolayer formation with 1:2 subcultivations took almost a month to occur. Morphologic changes in the cells began to appear over time, initially becoming fibroblastic (not shown), then later becoming more polygonal in appearance from approximately passage 35 onwards (Figure 1A, B). The final polygonal morphology was stable at passage 94 (Figure 1C). At the time of manuscript submission, the LMBG cells had been passaged 118 times. Complete monolayers had formed with 1:3 subcultivations in
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Development and Characterization of a Largemouth Bass Cell Line a
a
a
b
Rodman G. Getchell , Geoffrey H. Groocock , Emily R. Cornwell , Vanessa L. Schumacher , a
a
c
a
Lindsay I. Glasner , Barry J. Baker , Stephen A. Frattini , Gregory A. Wooster & Paul R. a
Bowser a
Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA b
Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
c
The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030, USA Published online: 21 Aug 2014.
To cite this article: Rodman G. Getchell, Geoffrey H. Groocock, Emily R. Cornwell, Vanessa L. Schumacher, Lindsay I. Glasner, Barry J. Baker, Stephen A. Frattini, Gregory A. Wooster & Paul R. Bowser (2014) Development and Characterization of a Largemouth Bass Cell Line, Journal of Aquatic Animal Health, 26:3, 194-201, DOI: 10.1080/08997659.2014.922517 To link to this article: http://dx.doi.org/10.1080/08997659.2014.922517
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:194–201, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.922517
ARTICLE
Development and Characterization of a Largemouth Bass Cell Line Rodman G. Getchell,* Geoffrey H. Groocock, and Emily R. Cornwell Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Vanessa L. Schumacher Downloaded by [Van Pelt and Opie Library] at 11:25 22 October 2014
Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
Lindsay I. Glasner and Barry J. Baker Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Stephen A. Frattini The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030, USA
Gregory A. Wooster and Paul R. Bowser Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 930 Campus Road, Ithaca, New York 14853, USA
Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22◦ C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week.
Cell lines provide an important diagnostic tool even though the availability of molecular assays is increasing. Close to 300 cell lines from finfish have been established since the first gonadal cell line was developed from Rainbow Trout Oncorhynchus mykiss (RTG-2; Wolf and Quimby 1962; Fryer and Lannan 1994; Lakra et al. 2011). During the last 20 years, 60% of the new cell lines have come from Asian investigators, where *Corresponding author: [email protected] Received February 18, 2014; accepted April 16, 2014
194
more than 80% of world aquaculture products originate (Lakra et al. 2011). As a result of its popularity as a game fish, Largemouth Bass Micropterus salmoides may now be the most widely introduced fish in North America (USDA 2006). Largemouth Bass are cultured at private, state, and federal facilities for recreational fishing and stocking programs. According to the 2005 Census of
Downloaded by [Van Pelt and Opie Library] at 11:25 22 October 2014
NEW LARGEMOUTH BASS CELL LINE
Aquaculture (USDA 2006), 192 U.S. fish farms reared Largemouth Bass, posting total sales of US$10.6 million that year. Largemouth Bass were introduced in China in 1983 (Deng et al. 2011) and have become widely cultured with annual production levels over 100,000 tons (Bai et al. 2008). Prior to 2011, the only significant viral pathogen of Largemouth Bass was the iridovirus, Largemouth Bass virus (LMBV), which had been isolated from Largemouth Bass in the eastern USA since 1991 (Plumb et al. 1996; Grizzle et al. 2002). But recently, three new viruses have been isolated from cultured Largemouth Bass in China, a ranavirus (Deng et al. 2011), a megalocytivirus (Ma et al. 2011), and a rhabdovirus (Ma et al. 2013). In Italy, a betanodavirus has been isolated from cultured Largemouth Bass (Bovo et al. 2011). The emergence of viral pathogens remains a significant constraint for aquaculture production and for the sustainability of biodiversity in the natural environment (Sahoo and Goodwin 2012). As viruses cannot reproduce on their own, the development of new fish cell lines aids in the detection and characterization of these emerging pathogens. Largemouth Bass virus has been isolated from 16 species of fish in the USA (Iwanowicz et al. 2013) and will replicate in several cell lines, and optimal cell culture methods for the detection of this virus have been determined (Piaskoski et al. 1999; McClenahan et al. 2005). Five cell lines previously tested for their LMBV susceptibility are: Bluegill fry (BF-2; ATCC certified cell line [CCL]-91), Fathead Minnow (ATCC CCL42), epithelioma papulosum cyprini (ATCC cell repository line [CRL]-2872), Channel Catfish ovary (ATCC CRL-2772), and Chinook Salmon embryo (CHSE-214; ATCC CRL-1681). All five of these lines have similar susceptibility to LMBV infection (McClenahan et al. 2005). The objective of this study was to establish and characterize a new cell line from the gonad of Largemouth Bass (LMBG) and evaluate the replication of LMBV in this cell line versus BF-2 cells.
METHODS Fish.—Largemouth Bass of various sizes were captured in June 2004 from Oneida Lake, New York, by electrofishing and transported to the Cornell University Aquatic Animal Health Program. Twenty Largemouth Bass were euthanized and the gonads were removed for processing and culture. This work was conducted under an animal protocol approved by the Institutional Animal Care and Use Committee of Cornell University. Cell line initiation.—Gonads from each of 20 adult Largemouth Bass were collected aseptically and processed separately. Following a procedure Bowser and Plumb (1980) developed to create an ovary cell line from Channel Catfish Ictalurus punctatus, Largemouth Bass tissues were minced and suspended in 20 mL of Hank’s balanced salt solution (HBSS), and the suspension was centrifuged at 1,500 × g for 5 min. The HBSS was then decanted and tissues were resuspended in 5 mL of 0.25% trypsin. The tissue suspension was immediately transferred to a diges-
195
tion flask and an additional 15 mL of 0.25% trypsin added. Cells were later harvested in aliquots after 8 h of digestion with continuous mixing. Harvested cells were washed and resuspended in phosphate-buffered saline (PBS) three times. Finally, cells were suspended in Hank’s minimal essential media (HMEM) with 20% fetal bovine serum (FBS) and 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine (PSG), and then aliquoted into primary 25-cm2 culture flasks. Flasks were incubated at room temperature, at approximately 22◦ C (hereafter 22◦ C). The cell lines were weaned onto HMEM with 10% FBS by passage 20. The flasks containing cells that adhered and grew were propagated. None of the flasks with testicular cells remained viable from the few males that were processed. For the initial 4–5 years, the LMBG cells were very slow growing. Complete monolayer formation with 1:2 subcultivations took almost a month to occur. Morphologic changes in the cells began to appear over time, initially becoming fibroblastic (not shown), then later becoming more polygonal in appearance from approximately passage 35 onwards (Figure 1A, B). The final polygonal morphology was stable at passage 94 (Figure 1C). At the time of manuscript submission, the LMBG cells had been passaged 118 times. Complete monolayers had formed with 1:3 subcultivations in
