Makrinos and Bowden 2016 b

Review

Growth characteristics of the intracellular pathogen,Piscirickettsia salmonis, in tissue culture and cell-freemedia

D L Makrinos and T J Bowden

School of Food and Agriculture, University of Maine, Orono, ME, USA

Abstract

Piscirickettsia salmonis is an intracellular bacteriumthat was first isolated and identified in fish cells.Several types of cell lines have been explored fortheir ability to provide the bacterium with a hostcell to replicate in. Tissue culture has been usedfor growth and cultivation for nearly two decades,until the facultative nature of P. salmonis was con-firmed upon the development of blood- andcysteine-based agar. Since then, research has con-tinued to drive the creation of novel agar andbroth formulations in order to improve the effi-cacy of cultivation of P. salmonis. Until now, thetechniques and components used for growth havenot been thoroughly discussed. In this review, themethods and formulations for growth ofP. salmonis in tissue culture and cell-free mediawill be examined.

Keywords: agar, broth, growth, intracellular, Piscir-ickettsia salmonis, tissue culture.

General introduction

Piscirickettsia salmonis, the causative agent of pis-cirickettsiosis or salmon rickettsia syndrome(SRS), is a Gram-negative bacterium first observedin Coho salmon (Oncorhynchus kisutch) in 1989

after an epizootic outbreak in Chile (Bravo &Campos 1989). It has been shown to cause mor-talities up to 90% and has since been identified inNorway, Scotland, Canada and the United States(Cvitanich, Garate & Smith 1991; Fryer &Hedrick 2003). The systemic pathogenesis anddifficulty to rapidly diagnose it has made piscirick-ettsiosis one of the biggest threats to the sustain-ability of Chilean salmonid aquaculture (Rozas &Enriquez 2014). Therefore, there is considerableinterest in the research behind the growth andinfectivity of P. salmonis.Piscirickettsia salmonis is a Gram-negative, intra-

cellular, generally non-motile, aerobic, non-encap-sulated, pleomorphic, but predominately coccoidbacterium with variable size ranging from 0.5 to1.5 lm (Fryer et al. 1990, 1992). It belongs tothe Gammaproteobacteria, which includes thegenera Francisella, Coxiella and Legionella (Fryer& Hedrick 2003). Other rickettsia including thegenera Rickettsia, Neorickettsia, Cowdria and Ehrli-chia are all grouped in the Alphaproteobacteria.The pathogenesis of P. salmonis can be charac-

terized by the systemic nature in various organsleading to the hallmark signs of anaemia andhaemorrhaging (Pulgar et al. 2015). Initially,P. salmonis uses a clathrin-dependent endocytosispathway to enter macrophages in vivo (Ramirez,Gomez & Marshall 2015). It infects macrophagesand has been shown to cause morphologicalchanges characteristic of apoptosis by methodssuch as chromatin condensation at the nuclearboundary (Rojas et al. 2010). P. salmonis

Correspondence D L Makrinos, School of Food and Agricul-

ture, University of Maine, 5735 Hitchner Hall, Orono, ME

04469-5735, USA (e-mail: [email protected])

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John Wiley & Sons Ltd

Journal of Fish Diseases 2016 doi:10.1111/jfd.12578

visualized by indirect fluorescence antibody testshas been seen to attach to the plasma membraneas early as 5 min p.i. (Smith et al. 2010).Although no surface projections have been seenin vitro, a previous study reported a ‘Piscirickettsiaattachment complex’ that facilitated attachment tothe ova in experimentally infected fish (Larenaset al. 2003). By 1 h p.i., P. salmonis has beenobserved inside the cytoplasm of rainbow troutmacrophages (Mccarthy et al. 2008). It replicatesby binary fission within membrane-bound cyto-plasmic vacuoles in fish cell lines or susceptiblehosts, leading to the characteristic cytopathic effect(CPE) observed in vitro (Fryer & Hedrick 2003).Replication using a budding mechanism has alsobeen reported to be common during propagationin vitro (Mccarthy et al. 2008).The preliminary diagnosis of piscirickettsiosis is

often performed by Gram-stained, Giemsa-stainedor methylene blue-stained kidney imprints orsmears (Fryer et al. 1990; Lannan & Fryer 1991).Furthermore, confirmation of the disease can bemade by serological methods such as fluorescentantibody tests and enzyme-linked immunosorbentassays, or by molecular assays such as PCR (Lannan& Fryer 1991; Mauel, Giovannoni & Fryer 1996;Marshall et al. 1998; Jamett et al. 2001).Piscirickettsia salmonis is predominately grown

in tissue culture to mimic natural in vivo condi-tions where it propagates within membrane-boundcytoplasmic vesicles. While tissue culture remainsthe common method for in vitro growth ofP. salmonis, continuous maintenance of cell lines,costly media and eukaryotic host cell debris pre-sent the need for alternative growth methods.These sources can be eliminated through cellpurification procedures or by the use of cell-freebacteriological cultures (Yuksel et al. 2006).Prophylactic measures for controlling the diseaseconsist of recombinant antigens and bacterins;however, higher cell densities in vaccines appearto improve efficacy (Marshall & Tobar 2014).Furthermore, optimization of a standard culturemedium would facilitate growth of high cell den-sities for vaccine development, broad studies onantibiotic susceptibility and ease of identificationand collection of isolates in the field.Growth of an intracellular bacterium in vitro

can be complex and presents problems whendetermining optimal growth conditions. In adefined growth environment, a bacterium willrequire particular essential nutrients, an optimum

temperature range, osmolality, presence or absenceof oxygen and carbon dioxide, light and a definedpH similar to in vivo conditions. By closely exam-ining these parameters, an understanding can begained about how this particular organism sur-vives, replicates and ultimately persists.Although studies have been published on vari-

ous bacteriological media for P. salmonis growth,these methods have not been extensively reviewed.Here, we sought to provide an overview of pub-lished techniques and discuss other similar intra-cellular bacteria which may provide insight forfuture growth studies.

Rickettsia-like Organisms (RLO’s)

Undescribed or uncharacterized intracellular fishpathogens are often designated as ‘Rickettsia-likeOrganisms’ (RLO). This is due to the inability toproperly diagnose and therefore categorize thebacterium at hand. During the initial identifica-tion of P. salmonis, uncertainty in isolation led toits characterization as an RLO (Fryer et al. 1990).It could be suggested that members of the orderRickettsiales have similar growth requirements dueto their phylogenetic relationship.The inability to culture RLO’s on bacteriological

media has been emphasized to limit the develop-ment of additional growth studies (Gollas-Galv�anet al. 2014). Breaking this growth barrier wouldallow studies to focus more on the pathogenesis andtherapeutic control measures rather than centeringthe attention on growth requirements.

Intracellular organisms

When analysing the in vitro growth requirementsof P. salmonis, closely related intracellular bacteriasuch as Legionella pneumophilia, Coxiella burnetiiand Renibacterium salmoninarum could providethe groundwork to identify essential media com-ponents. Intracellular bacteria grow in a particularniche within target host cells; therefore, it isimportant to identify a metabolically permissivebuffer with a pKa near the predicted pH of thebacterium’s ideal growth environment (Omslandet al. 2008). In the case of C. burnetii, the strongbuffering capacity of citrate buffer facilitated thepH range of 4.5–5.0, which increased metabolicactivity through incorporation of [35S] cysteine–methionine as measured by protein synthesis usingscintillation counting and autoradiography.

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Journal of Fish Diseases 2016 D L Makrinos & T J Bowden In vitro growth of P. salmonis

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Casamino acids and L-cysteine have displayed anadditive effect on metabolic fitness using the samemethods as above; however, no bacterial replica-tion was observed, indicating that metabolic fit-ness is not necessarily indicative of replication(Omsland et al. 2009; Omsland, Hackstadt &Heinzen 2013). An appropriate pH should be uti-lized in artificial growth media to mimic host cellcytoplasmic vacuoles.The Gram-positive intracellular bacterium Reni-

bacterium salmoninarum is most commonly grownon KDM-2 medium, which contains peptone, yeastextract, L-cysteine and 20% foetal bovine serum(FBS), which are already found in P. salmonis media(Evelyn 1977). R. salmoninarum has also been grownon a charcoal agar (KDM-C; 0.05–0.2%), whicheffectively replaces serum in media (Daly & Steven-son 1985). Starch and skim milk powder have alsoreplaced serum in this media, but not to the successof charcoal. Media such as KDM-C provides aserum-free alternative, which could be critical due tothe fact that many intracellular bacteriological mediacontain serum. Charcoal has been successfully incor-porated into the media of other bacteria includingLegionella spp., Bordetella spp. and Leptospira spp(Daly et al. 1985). R. salmoninarum grown on aselective agar medium displayed a prolonged lagphase, taking up to 6 weeks for macroscopic coloniesto appear (Benediktsdottir, Helgason & Gudmunds-dottir 1991). Although it was previously believed tobe a strict aerobe, R. salmoninarum grown in micro-aerophilic conditions has been shown to increase theoverall size (diameter) of individual colonies(Hirvel€a-Koski 2008). Growth on KDM2 agar-basedmedia requires incubation at 16 °C for 5–12 weeks.For R. salmoninarum, a prolonged incubation periodis necessary due to a delayed growth physiology.When growing L. pneumophila in liquid media,

L-cysteine was found to be essential for growth(100 lg mL�1), while the supplementation ofiron was not (Barker, Farrell & Hutchison 1986).Among similar intracellular organisms, compo-nents such as L-cysteine and serum appear to becommon throughout media. L-cysteine is com-monly added in abundance to media, because it isthe oxidized form of L-cystine, which containstwo cysteine molecules joined together, making itdifficult for L. pneumophilia to utilize cystine(Ewann & Hoffman 2006). Iron was suggested tocatalyse interconversion of cysteine forms andtherefore thought to be an essential component ingrowth. Authors found increased growth and

uptake of L-cysteine at a pH of 6.2 compared to6.9, which is similar to partly acidified phago-somes, where L. pneumophilia replicates in macro-phages. Additionally, these pH ranges couldfurther support the notion of how P. salmonisgrows in vitro. Similar intracellular bacteria suchas L. pneumophilia, C. burnettii and R. salmoni-narum cultured in cell-free systems could provideinsight on critical media components.

Tissue culture

Many intracellular bacteria require a host cell topropagate within. The most common approachfor re-isolation and growth of P. salmonis is incultured fish cells using tissue homogenate (Fryeret al. 1990; Lannan & Fryer 1991). The tissues ofchoice for isolation are often kidney, liver andblood during an active infection; however, thebrain has also been included as a tissue for detect-ing the pathogen (Lannan & Fryer 1991; Skar-meta et al. 2000). P. salmonis is sensitive to manyantibiotics often used for in vitro cell culture isola-tions; therefore, antibiotic compounds are com-monly excluded from culture medium leavingcells vulnerable to other forms of contaminationwhen inoculating with infected fish tissue (Lannan& Fryer 1991; Almendras & Fuentealba 1997).The type strain of P. salmonis LF-89 (ATCC

VR-1361) was first isolated in chinook salmonembryo cells (CHSE-214) in 1989 by placinginfected kidney tissue onto a cell monolayer (Lan-nan, Winton & Fryer 1984; Fryer et al. 1990).For proper isolation of the pathogen from infectedfish, tissue was aseptically removed and homoge-nized at 1:20 in antibiotic-free balanced salt solu-tion (BSS) or culture media (Lannan & Fryer1991). It should be further diluted 10�2 and10�3 w/v, before inoculation onto a cell culturemonolayer. When cells are infected, they begin toswell up and appear as clusters of rounded cellsand plaque-like zones of clearance producing acytopathic effect (CPE), ultimately leading to lysisand destruction of the entire cell layer (Fig. 1).Depending on the infectious dose, cells will showCPE after 5–10 days and growth is retarded attemperatures below 10 °C and above 21 °C lead-ing to delayed infectivity (Fryer et al. 1990;Cvitanich et al. 1991).

Cell lines used for cultivation of P. salmonis. Dur-ing the initial outbreaks of SRS in Chile, a

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rickettsial-like organism was the first observedpathogen in an aquatic poikilotherm from theorder Rickettsiales. Although the CHSE-214 cellline was used for the initial isolation, the authorsalso reported the use of coho salmon embryo(CSE-119), chum salmon heart (CHH-1) andrainbow trout gonad (RTG-2) as salmonid-derivedcells (Table 1). Warm water fish cells includingEpithelioma papulosum cyprini (EPC), fatheadminnow caudal fin (FHM), brown bullhead poste-rior trunk (BB) and bluegill fry caudal trunk (BF-2) were also cultured to explore their susceptibilityto the pathogen. The only two cell lines that didnot produce CPE were BB and BF-2. CPE wasnot observed in cell cultures containing antibi-otics; however, CPE appeared after 10 days ofincubation at 15 °C in antibiotic-free CHSE-214cultures. Transferring infected medium to na€ıvecell monolayers caused CPE after 5–6 days at 15or 18 °C, and the entire cell monolayer was lysedafter 14 days (Fryer et al. 1990).Soon after, a rickettsia-like organism (RLO)

causing the proposed disease ‘Salmonid RickettsialSepticaemia’ was investigated in various types ofculture media (Cvitanich et al. 1991). Authorsattempted isolation in CHSE-214, CHH-1, CSE-119, RTG-2, salmonid-derived cell lines as well asEPC, FHM, BB and BF-2 warm water species celllines in antibiotic-free MEM-10 by inoculatingcultures with homogenized kidney tissue dilutedin balanced salt solution (BSS) for 30 days at15 °C (Cvitanich et al. 1991). CPE was observedbetween 7–21 days. This RLO did not induceCPE in the BB and BF-2 cell lines (Fryer et al.

1990; Cvitanich et al. 1991). The use of a rangeof different cell lines in comparative studies couldindicate whether the organism is a RLO or Piscir-ickettsia-like organism (PLO).More recently, studies on P. salmonis have used

different fish cell lines such as Atlantic salmonkidney (ASK; Smith et al. 2015), rainbow troutspleen (RTS-11) and Atlantic salmon head kidney(SHK-1; Ramirez et al. 2015), while alternativecell lines such as Spodoptera frugiperda (SF-21;Birkbeck et al. 2004) have also been examined.SF-21, derived from the fall armyworm, is aninsect cell line which enables cultivation ofP. salmonis at higher titres than the CHSE-214cell, and therefore, it has been proposed that aninvertebrate or non-fish poikilotherm vector maybe present (Birkbeck et al. 2004). This cell line isadvantageous, because it can yield the highest celldensities of P.salmonis enabling bacterin vaccinesto increase antigenic content.During an active infection, P. salmonis is found

in the kidney and spleen of fish; therefore, SHK-1and RTS-11 cells are believed to be appropriate tomimic in vivo macrophage-like properties (Rojaset al. 2010; Yanez et al. 2012). P. salmonisactively targets phagocytic cells and induces apop-tosis unlike epithelial cells, making macrophage-like cell lines a more natural host to studyinfectivity.Cell concentrations in tissue culture are usually

represented by a TCID50 or plaque assay titrationin which the countable CPE or plaques presentare enumerated. P. salmonis generally replicates totitres of 106 and 107 TCID50 mL�1 in fish cell

(a) (b)

Figure 1 A na€ıve CHSE-214 cell monolayer (a) and Piscirickettsia salmonis LF-89 inducing cytopathic effect (CPE) and plaque for-

mation in a CHSE-214 cell monolayer at day 10 post-infection at 109 magnification (b). [Colour figure can be viewed at

wileyonlinelibrary.com]

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cultures (Fryer & Mauel 1997). A single freeze–thaw from �70 °C has been shown to decreasetitres by 99%; however, the addition of 10%dimethyl sulphoxide (DMSO) enables cryopreser-vation (Fryer & Lannan 1996). Titrations couldbe further evaluated to determine which condi-tions appear most suitable for optimal growth.

Cell-free media

Until 2008, it was thought that P. salmonis couldnot grow on cell-free bacteriological media (Fryeret al. 1990; Cvitanich et al. 1991; Fryer &Hedrick 2003). Cell culture enabled consistentgrowth, but the need for cultures without host cellcontamination led researchers to look further foralternatives. A breakthrough occurred when twotypes of agar formulations were reported enablingroutine cell-free cultivation (Mauel, Ware &Smith 2008; Mikalsen et al. 2008). Subsequently,researchers found additional formulations of solidand liquid media ideal for P. salmonis (G�omez,Henr�ıquez & Marshall 2009; Marshall et al.2012; Yanez et al. 2012, 2013). With variousmethods published, commonality among formula-tions can be evaluated to formulate a highly effica-cious medium for growth.

Agar (solid media)

Solid media or agar was explored after the firstoutbreaks in Chile occurred to examine thegrowth requirements of the pathogen (Fryer et al.1990; Cvitanich et al. 1991). To provide thereader with an extensive background of methods

previously used, all attempted formulations will bereviewed in the order they were published.Agar culture of intracellular bacteria presents a

key advantage to analyses by eliminating the needfor a host cell to grow in. However, this switch ingrowth environments can affect the organism andits physiological status. In tissue culture, titrationsbased on a TCID50 provide an estimation of thenumber of virulent bacteria present causing CPE.By growing P. salmonis on agar, a more accuratecell count can be achieved. Colony-forming units(CFU) are a common method to report the num-ber of viable bacterial cells grown on agar.Although this method is reliable for many otherbacterial pathogens, it is not necessarily pre-dictable for all organisms. Cell counts on a hemo-cytometer are another method used to report cellcounts, but due to the small size of P. salmonis,accurate estimations are still jeopardized. A Taq-Man qPCR assay has been utilized to quantifyP. salmonis in cell culture and bacteriologicalmedia; however, PCR-based assays detect onlynucleic acids and therefore do not indicate cellviability (Corbeil, Mccoll & Crane 2003). Here,we will discuss the different bacteriological mediaformerly attempted for culture of P. salmonis. Byreporting methods that are both successful andunsuccessful, the reader may obtain additionalinformation not previously observed.Initially, researchers attempted culture in media

including tryptic soy agar, tryptic soy agar with5% coho salmon blood, fluid thioglycolate, OFmedium with 1% dextrose, MEM-10, Dorset eggmedium, Petragnani medium, Herrold’s egg yolkagar without malachite green, KDM-2 broth with

Table 1 Cell lines previously tested for growth of Piscirickettsia salmonis

Cell line Species of origin CPE observed Primary source

CHSE-214 (ATCC CRL 1681) Chinook salmon embryo Oncorhynchus tshawytscha

(Walbaum)

Yes Fryer et al. (1990)

CSE-119 (ECACC 95122019) Coho salmon embryo Oncorhynchus kisutch Yes Fryer et al. (1990)

CHH-1 (ATCC CRL 1680) Chum salmon heart Oncorhynchus keta (Walbaum) Yes Fryer et al. (1990)

RTG-2 (ATCC CCL 55) Rainbow trout gonad Oncorhynchus mykiss Yes Fryer et al. (1990)

EPC (ECACC 93120820) Epithelioma papulosum cyprinid or Common carp

Cyprinus carpio (Linnaeus)

Yes Fryer et al. (1990)

FHM (ATCC CCL 42) Fathead minnow Pimephales promelas (Rafinesque) Yes Fryer et al. (1990)

BF-2 (ATCC CCL 91) Bluegill fry caudal trunk Lepomis macrochirus

(Rafinesque)

No Fryer et al. (1990)

BB (ATCC CCL 59) Brown bullhead Ictalurus nebulosus (Lesueur) No Fryer et al. (1990)

SHK-1 (ECACC 97111106) Atlantic Salmon Salmo salar Head Kidney Yes Yanez et al. (2012)

ASK (ATCC CRL 2747) Atlantic salmon Kidney Yes Smith et al. (2015)

RTS-11 (ATCC CRL 2797 Rainbow trout spleen (ATCC CRL 2797) Yes Rojas et al. (2010)

SF-21 (ECACC 05022801) Fall armyworm ovary Spodoptera frugiperda Yes Birkbeck et al. (2004)

XTC-2 (ATCC CCL 102) African clawed frog kidney Xenopus laevis N/A Birkbeck et al. (2004)

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10% FBS, charcoal agar and mycoplasma med-ium, which were inoculated with previouslyinfected cultures with extensive CPE (Fryer et al.1990). The media was then incubated for 30 daysat 15 °C and observed for growth. No growthwas observed on any of the bacteriological media.The use of one incubation temperature at 15 °Cmay have limited the ability of the particular iso-late to grow.Another study tested inoculation techniques

using agar, bottles, tubes and broth with 0.1 mLof 10�2, 10�3 and 10�4 dilutions of infected kid-ney tissues containing 107–108 RLO g�1

(Cvitanich et al. 1991). The media tested wereAeromonas selective agar with 5% sheep blood(SB), Mycoplasma medium, brain–heart infusionagar with 5% SB, Columbia broth with CO2 andsodium polyanethol sulphonate (SPS), DNASEagar tubes, egg yolk agar, Gram-negative broth,Haemophilus-selective agar, heart infusion brothwith 1% dextrose, Loeffler medium tubes, Mid-dlebrook 7H11 agar, Mueller-Hinton agar(MHA), MHA with 5% SB, Mueller-Hintonchocolate agar, nutrient gelatin tubes, OF mediumwith 1% dextrose, Regan-Lowe Pertussis agar,Sabouraud dextrose agar bottles, thioglycollatemedium (Thio) without indicator (with CO2,SPSm and enrichment), Thio with NaHCO3 andyeast extract, Thio with resazurin and CO2, tryp-tic soy agar (TSA) with 5% SB, tryptic soy brothwith CO2 and SPS, Lowenstein–Jensen mediumtubes and mycoplasma agar/broth. Dehydratedmedia including nutrient agar, BHIA, standardmethods agar, TSA, TSA+ with 1% InstantOcean (IO) and tryptone (1%) dextrose (0.33%)yeast extract (0.5%) broth with 1% IO were eval-uated. Lastly, Cytophaga agar with and without1% IO, charcoal agar and broth, medium 199with Hank’s salts and 10% FBS adjusted to pH7.4, MEM-10 and MEM-10 conditioned mediumfrom a 14-day CHSE-214 cell culture which was0.45-lm filter-sterilized to remove any non-adher-ent cells. The inoculated media in Petri disheswere placed in plastic bags and either incubatedaerobically or with increased CO2. For the mediain tubes or bottles, cultures were incubated aerobi-cally, while strict anaerobic conditions were usedfor media containing thioglycollate. The mediatested were incubated at 14–15 °C for 8 weeks,and the TSA, TSA+, MHA, BHIA and BA werethe most routinely attempted and additionallyincubated aerobically at 27 °C. Of the 41 diverse

culture media tested, all failed to grow the organ-ism from infected kidney tissue homogenate.Piscirickettsia salmonis was commonly thought

to be an obligate intracellular pathogen until thefacultative nature of the pathogen was confirmedwhen it was first cultivated on cysteine-enrichedcell-free media (Mauel et al. 2008; Mikalsen et al.2008). Infected cell cultures from previouslyinfected fish were streaked onto three types of agarmedia including blood agar (BA), BA with 2%NaCl (BAS) and Cystine Heart Agar (CHAB)containing 25 g L�1 Bacto Heart Infusion broth,10 g L�1 glucose, 1 g L�1 L-cysteine, 15 g L�1

agar and 2 g L�1 haemoglobin supplemented with5% sheep blood (Table 2; Mikalsen et al. 2008).Plates were incubated for 14 days at 22 °C (BAand CHAB) and 15 °C (BAS), respectively. Ini-tially, pinpoint colonies (0.1 mm) were observedon CHAB cultures after 4–5 days of incubationfollowed by more distinct colonies, which weredescribed as slightly convex, grey–white, shiny andcentrally opaque with translucent, slightly undu-lating margins (Fig. 2). Although the subculturewas successful on CHAB, the BA and BAS platesdid not produce visible colonies after 14 days.Aerobic cultures were also incubated at 4, 10, 15and 30 °C. After 5 days at 10 °C, growth wasobserved, while 22 °C (+5% CO2) was found toretard growth. CHAB colonies were positivelyidentified by PCR against the 16S and ITS regionand sequenced with 99% identity to P. salmonisLF-89 type strain.Serial passages of P. salmonis in vitro raise the

question whether or not repetitive passages lead toa decrease in in vivo virulence or a general physio-logical alteration. To test this hypothesis, 200 g ofAtlantic salmon held in sea water at 12 °C was IPinjected with CHAB-cultivated cells that had beenpassaged five times (Mikalsen et al. 2008). Tenfish per group were injected with 3 9 107 CFU,3 9 105 CFU and 3 9 103 CFU as well as acohabitant group which was injected with 0.9%saline. After 30 days, cumulative mortality washighest in the 3 9 107 group with 70% and only20% in 3 9 105. Koch’s Postulates were demon-strated when P. salmonis was subsequently re-iso-lated dead fish. The pathogenesis of P. salmonis ininfectious challenge trials is slow to develop andchallenges have been carried out for a period upto 50 days; therefore, these findings are not con-clusive (Wilhelm et al. 2006; Tobar et al. 2011).The authors explain that agar isolation did not

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lead to a sudden drastic loss of virulence; however,none of the cohabitants died during the challengeperiod. No cohabitee fish were injected with cellculture supernatant to compare virulence to agarcolonies; thus, additional research could be con-ducted to determine virulence of P. salmonisgrown in alternative media.A new report soon after used infected cell cul-

ture supernatant to inoculate BFCG agar, whichcontains 5% sheep blood, 3% FBS, 0.1% cysteineand 1% glucose (Table 2; Mauel et al. 2008).After 6 days of incubation at 16 °C, small whitecolonies (0.1 mm) with convex circular morphol-ogy became visible. Agar cultures were also madewithout the addition of FBS (BCG) and withoutcysteine (BFG) to determine which componentsmay be essential for growth. Similar colonies wereidentified at 6 days on BCG plates; however, after3 weeks, no growth was visible on BFG plates,

making it apparent that the addition of cysteinewas essential for growth of P. salmonis.Growth of P. salmonis is relatively slow and on

solid media can take 4–8 days before visible colo-nies are present. This could explain why growth isoften reported in absorbance units or cell numbersto facilitate rapid enumeration. AlthoughP. salmonis grew on CHAB, the inability to effec-tively recover viable bacteria from all media dueto considerable variations in growth rates makes itchallenging to determine cell concentrations(Yanez et al. 2012). P. salmonis may retain itsinfectivity; however, the switch between cell-freeenvironments could potentially be inhibitory togrowth as a consequence of a lack of optimalconditions.A follow-up study compared the growth of

P. salmonis on CHAB agar (Mikalsen et al. 2008)to two new versions of trypticase soy agar called

Table 2 The optimum growth temperature and iron/blood components of published agar media

Reference Media name Optimum temperature (°C) Iron/Blood components

Mikalsen et al. (2008) CHAB Agar 22 5% sheep blood

2 g L�1 haemoglobin

Mauel et al. (2008) BFCG Agar 16 3% FBS

5% Sheep blood

Vera et al. (2012) Vera Agar 17 2.5% FBS

10 mg L�1 ferric chloride

Yanez et al. (2013) Austral-TSFe and

TSHem Agar

18 5% FBS

5% (2% haemoglobin) or

0.05 mM ferric nitrate

Otterlei et al. (2016) SRS-BA Agar 16–19 and 19–22 5% sheep blood

5% FBS

0.2 mM ferric nitrate

(a) (b)

Figure 2 Growth of Piscirickettsia salmonis LF-89 on CHAB agar at 22 °C (a) and TSHem agar at 18 °C (b). [Colour figure can

be viewed at wileyonlinelibrary.com]

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Austral-TSFe and Austral-TSHem, with varyingconcentrations of L-cysteine, sodium chloride andferric nitrate (Yanez et al. 2013). To begingrowth, 0.1 mL of frozen cell culture supernatantswere spread over the surface of plates and incu-bated at 18 °C for 15 days (Fig. 2). Previously,stock cultures were made by resuspending coloniesin 1 mL of L-15 containing 20% FBS and 10%DMSO at �80 °C. To quantify bacterial growth,colonies were collected, resuspended in L-15 andOD600 was recorded. Visible, white, pinpointcolonies were observed after 8–10 days. Althoughthere was no significant difference in growth bio-mass between the three media (Austral-TSFe,TSHem and CHAB), Austral-TSHem displayedan absorbance of 13.6% higher than CHAB. Theauthors selected concentrations of L-cysteine,sodium chloride and ferric nitrate at 0.5 g L1,7.5 g L1 and 0.20 mM respectively, based onCFU mL�1 and OD600. The authors noted thatno growth was observed in agar formulations(Austral-TSFe) without cysteine or iron (data notshown); therefore, both constituents are believedto be essential for growth. Ferric nitrate and BDBBLTM Haemoglobin Solution were effective alter-natives to blood. However, this is the first reportthat indicated iron is necessary for growth ofP. salmonis (Yanez et al. 2013).A study was recently published comparing phe-

notypic variations of P. salmonis isolates grown onvarious agar media (Otterlei et al. 2016). A newoptimized growth media called SRS blood agar(SRS-BA) was developed using 40 g L�1 TSA,20 g L�1 Red Sea Salt (RSS), 50 mL sheepblood, 1 g L�1 of L-cysteine, 5 g of D-glucose,50 mL FBS, 0.2 mM ferric nitrate and reverseosmosis water to a final volume of 1000 mL(Table 2). The authors note that RSS was supple-mented in the media to simulate the natural sea-water composition. To test the different growthmedia, two colonies of each P. salmonis isolatewere streaked onto SRS-BA, Austral-TSHem,CHAB, CHAB with 0.2 mM Fe, blood agar (BA),BA with 2% NaCl, marine agar (MA) and tryp-tone–yeast extract salts with glucose agar (FLPA)and incubated at 19 °C for 14 days. No growthwas observed on MA and FLPA media; however,certain isolates appeared to have strict growthrequirements, which were differentiated as ‘fastidi-ous’ (16–19 °C) and ‘less fastidious’ (19–22 °C).Comparison of various culture media indicatedthat SRS-BA was the ideal medium for cultivation

of the isolates studied. This study highlighted animportant concept that not all P. salmonis isolatesgrow in the same way. Optimum temperaturesclearly vary, and when developing a new cell-freemedium, various geographical isolates should beanalysed.Being one of the first media published, the

CHAB agar is widely cited and accepted for cell-free growth of P. salmonis (Mikalsen et al. 2008).The ability to grow at room temperature (22–23 °C) offers a distinct advantage without the needfor additional incubation. CHAB agar has also beenused in comparative studies to further test novelagar formulations (Yanez et al. 2013; Otterlei et al.2016). The use of sheep blood and haemoglobin asiron sources has since been explored in other for-mulations (Yanez et al. 2013). Previously, it wasreported that P. salmonis grew in the absence ofFBS on BCG agar (Mauel et al. 2008). Compo-nents such as FBS and sheep blood make definingspecific nutrient requirements difficult, because ofthe lack of knowledge on what these sources arespecifically composed of. The use of FBS as a sup-plement in cell culture as well as agar and broth cul-ture could be potentially alleviating the metabolicshift between eukaryotic cell environments, to acell-free growth environment. Although divergingaway from partially undefined iron sources seemspractical, it is often challenging to find suitablereplacements. It has been suggested that intactblood cells may be essential to efficient P. salmonisdevelopment and multiplication (G�omez et al.2009). Blood-free media compositions have beensuccessful in growth and used in other studies(Henriquez et al. 2013). These compositions con-tain ferric chloride in replacement of blood andserum components supporting the notion that aniron source is essential for growth of P. salmonis.

Broth (liquid media)

Broth or liquid media can play an advantageousrole in the rapid growth and production of bacte-rial cultures. After the first reports of novel agarfor P. salmonis, new broth cultures followed soonafter. We previously covered unsuccessful liquidmedia attempted after the first isolation ofP. salmonis; hereafter, we will address successfulformulations for growth in liquid media (Fryeret al. 1990; Cvitanich et al. 1991).The authors of CHAB agar also reported the

development of a cysteine heart broth with and

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without 5% sheep blood and found it could sup-port growth of Norwegian isolate 5692 whenincubated at 22 °C with gentle shaking. Overallgrowth rates and performance were not reportedmaking comparisons challenging (Mikalsen et al.2008).A liquid medium Blood Broth (BB) containing

10 g L�1 tryptone, 2 g L�1 peptone, 2 g L�1

yeast extract, 5 g L�1 NaCl, 0.1% L-cysteine and5% of fish blood lysate (v/v) from rainbow troutwas formulated for growth of P. salmonis(Table 4; G�omez et al. 2009). To prepare rain-bow trout lysate, whole blood was sonicated at 11root mean square (RMS), centrifuged at11 000 rpm for 30 min, and the resulting super-natant was filtered through 0.22-lm filters andstored at 4 °C. Fish blood lysate presents a uniqueblood component containing cytoplasmic cell con-tents to simulate a more natural growth environ-ment. Using BFCG agar media previouslydescribed (Mauel et al. 2008), a single colonyfrom BFCG agar was resuspended into 500 lL ofPBS and 50 lL was used to inoculate 5 mL ofBB. The cultures were incubated with gentle shak-ing for 14 days at 17 °C, and the OD620 wastaken every 24 h to construct a growth curve.Growth reached a maximum OD620 of 0.25 after13 days and was confirmed by inoculating BFCGcolonies in CHSE-214 and RTS11 cells to observefor presence of CPE (Table 3). This was the firstreport of a detailed cell-free liquid media forP. salmonis, which they suggest is possibly due toits dependency on eukaryotic blood cells todivide.Biofilm production by P. salmonis was compared

in a liquid medium called MC1 containing10 g L�1 peptone, 10 g L�1 yeast extract,

10 g L�1 NaCl, 1% glucose, 0.1% L-cysteine,0.2 g L�1 K2HPO4, 0.15 g L�1 MgSO4,0.4 g L�1 KCl and 10% FBS to marine broth(MB) when incubated at 23 °C at 100 rpm(Table 4; Marshall et al. 2012). Broth cultures wereinitially inoculated with a single colony from modi-fied BFCG agar grown at 23 °C (Mauel et al.2008). Using MC1 media, authors were able toconduct fluorescence microscopy and SEM ofP. salmonis in a growth environment outside of ahost cell not previously examined. The commonlyused Luria-Bertani broth (LB), which induces bio-film formation in V. cholera, was compared toMC1 broth, because both are nutrient rich. How-ever, the LB medium did not support growth ofP. salmonis. Marine broth (MB) was chosen as anutrient-limited broth to simulate harsh environ-mental conditions, therefore stimulating biofilmaggregation. It was clear that the high salt and lim-ited nutrient content of the MB broth caused thebacterium to aggregate more rapidly in biofilmssimilar to the natural marine environment.A study screening 110 specific marine and tryp-

tone soy broth formulations supplemented withL-cysteine and ferric nitrate found only two broths(Austral-SRS and TSFe) that enabled growth ofthe bacterium (Yanez et al. 2012). For furtherevaluation, the use of several combinations/con-centrations of marine broth, sodium chloride,tryptone soy, Yeastolate, L-cysteine, ferric salt andhaemoglobin was evaluated. The two broth mediasuccessful in growth were further examined in anindependent study to determine the optimal con-centrations of L-cysteine (1–50 g L�1) and ferricnitrate (0.001–1 mM). The Austral-SRS broth(20 mL) generated the best growth when inocu-lated with colonies from agar plates, reaching anOD600 of 1.8 after 10 days of incubation at18 °C with gentle agitation (50 rpm) at 0:24 dark(Table 3). Interestingly, the authors reported themeasure of growth in CFU mL�1 by directmicroscopic count, but observed poorly definedcolonies that lead to inaccuracies in estimations.A liquid growth media was also reported to

reach an OD600 of 1.8 after 12 days when grownat 17 °C (Table 4; Vera et al. 2012). The authorsused the broth culture to formulate an agar mediawhich also supported growth, displaying distinctcoccid morphology after 12 days of incubation at17 °C.Another study optimized growth of P. salmonis

in a novel liquid medium free of blood and serum

Table 3 The optimum growth temperatures and maximum

optical density of Piscirickettsia salmonis obtained in liquid

media

Reference Media name

Optimum

temperature

(°C)Maximum optical

density (600–620)

Mikalsen

et al. (2008)

CHB 22 Not reported

G�omez

et al. (2009)

BB 17 0.25 after 13 days

Vera

et al. (2012)

Vera 17 1.8 after 12 days

Yanez

et al. (2012)

Austral-SRS 18 1.8 after 6 days

Henriquez

et al. (2013)

BM 23–27 1.7 after 37.5 h

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(BM 1–4) with varying amounts of yeast extract,peptone, glucose, glutamic acid and ammoniumsulphate (Henriquez et al. 2013). As a replacementto blood- and/or serum-based components, theauthors use iron (II) sulphate heptahydrate instead.The core components of BM broth consist of2.0 g L�1 yeast extract, 2.0 g L�1 peptone frommeat, 1.32 g L�1 ammonium sulphate, 0.1 g L�1

magnesium sulphate, 6.3 g L�1 dipotassium phos-phate, 9.0 g L�1 sodium chloride, 0.08 g L�1 cal-cium chloride and 0.02 g L�1 iron (II) sulphate(Table 4). There are four different compositionsto the BM broth, and over time, the authors opti-mized particular constituents for each. BM brothdiffers from BM3 in that it contains ammoniumsulphate and does not contain glutamic acid. Thehighest biomass concentration was reached in theBM3 broth after 37.5 h at an OD600 of 1.7,which was faster than previously reported(Table 3; Yanez et al. 2012). P. salmonis grown inBM4 broth and inoculated in CHSE-214 cellsremained pathogenic, confirming similar results toprevious studies (G�omez et al. 2009; Marshallet al. 2012). The BM broth is advancement tocell-free growth, because not only does it growwithout blood or FBS, but it is also the onlymedia reported to grow without the addition ofL-cysteine.The generation time of P. salmonis in cell-free

media is believed to be 5–7 h (Marshall et al.2012). Furthermore, P. salmonis grown in liquidmedia (BM3) has been shown to have a genera-tion time between 4.62 and 5.33 h (Henriquezet al. 2013).

Antibiotic susceptibility is difficult to performwithout a reliable P. salmonis-specific culturemedia. Austral-SRS broth has been shown to be asuitable alternative to Mueller-Hinton mediumwhen determining minimum inhibitory concentra-tions (MIC) of P. salmonis antibiotic susceptibility(Yanez et al. 2014). Additionally, SRS-BA hasenabled testing of antibiotic susceptibility, findingthat all isolates studied were sensitive to oxytetra-cycline and florfenicol, which yielded the largestzones of inhibition.Liquid cultivation of P. salmonis is well-estab-

lished and offers an inexpensive means for growth.As various types of nutrient-rich media continueto be published, research has begun to uncovermore details on the complex nature of this intra-cellular pathogen.

Temperature

The role of temperature in bacteriological cultureis a pivotal factor in understanding growth kinet-ics. The maximum temperature range for agar hasbeen defined between 8 and 25 °C for SRS-BA(Otterlei et al. 2016), while liquid culture cansupport growth between 17 and 29 °C (Hen-riquez et al. 2013). Growth was achieved in BM1broth between 17 and 29 °C, with an optimumtemperature range between 23 and 27 °C. Fastidi-ous isolates, which tend to have similar genotypes,grow optimally on agar between 16 and 19 °C,whereas less fastidious isolates grow best between19 and 22 °C (Otterlei et al. 2016). A previousstudy observed optimal agar growth at 22 °C

Table 4 An overview of the components in BB, Austral-TSFe, BM3, MC1 and Vera broth

Component

BB (G�omez

et al. 2009)

MC1 (Marshall

et al. 2012)

Vera (Vera

et al. 2012)

Austral-TSFe

(Yanez et al. 2013)

BM3 (Henriquez

et al. 2013)

FBS N/A 10% 2.5% 5% N/A

Fish blood Lysate 5% N/A N/A N/A N/A

D-glucose N/A 10 g L�1 2.5 g L�1 5 g L�1 5 g L�1

L-cysteine 0.1% 0.1% 0.1% 0.5% N/A

Ferric nitrate N/A N/A N/A 0.2 mM N/A

Ferric (II) sulphate N/A N/A N/A N/A 0.02 g L�1

Ferric chloride N/A N/A 0.01 g L�1 N/A N/A

Tryptone Soy Broth 10 g L�1 N/A N/A 36 g L�1 N/A

Peptone from casein N/A 10 g L�1 17 g L�1 N/A 8 g L�1

Dipotassium phosphate N/A 0.2 g L�1 2.5 g L�1 N/A 6.3 g L�1

Sodium chloride 5 g L�1 10 g L�1 20.0 g L�1 7.5 g L�1 9.0 g L�1

Potassium chloride N/A 0.4 g L�1 N/A N/A N/A

Magnesium sulphate N/A 0.31 g L�1 N/A N/A 0.1 g L�1

Calcium chloride N/A N/A N/A N/A 0.08 g L�1

Yeast extract 2 g L�1 10 g L�1 N/A N/A 8.0 g L�1

Glutamic acid N/A N/A N/A N/A 2.0 g L�1

Soy peptone N/A N/A 3.0 g L�1 N/A N/A

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(Mikalsen et al. 2008), while P. salmonis was latercultivated on agar and in broth between 16 and17 °C (Mauel et al. 2008; G�omez et al. 2009).While the LF-89-type strain remains the referencestandard for most studies, new geographic isolatesfrom Chile, Norway, Canada and Scotland con-tinue to generate interest due to the emergence ofvariable strains, which display different in vitrogrowth kinetics and antibiotic susceptibility.

pH

The pH of a defined medium plays a critical rolein the metabolic requirements of a particular bac-terium. In vivo, P. salmonis propagates within hostcell vacuoles where the pH range is likely moreacidic than the external environment. Despite aparticular pH niche being essential, little informa-tion is discussed on the optimum pH range forgrowth of this intracellular organism. While fewstudies report the pH of P. salmonis agar media,SRS-BA is known to have a final pH of6.8 � 0.2 (Otterlei et al. 2016). For Austral-SRSbroth, a final pH between 6.0 and 6.4 was usedfor successful growth (Yanez et al. 2012). Onestudy in particular found similar growth patternsat a pH of 6.6 and 7.1 in broth culture(Henriquez et al. 2013). The authors report it issimilar to the range of another intracellular bacteria,Legionella pneumophilia, which can grow at a pHof 6.9 and optimally at 6.5 (Barker et al. 1986).Surprisingly in this study, the pH during growthincreased rather than decreased; therefore, a finalpH of 6.6 was chosen for further investigation.This increase has been suggested to be due to thedegradation of amino acids present in peptoneand yeast extract (Henriquez et al. 2013). Addi-tional research on permissive buffers ideal for theoptimal pH range of P. salmonis could facilitateincreased growth.

Conclusion

Currently, in vitro growth of P. salmonis is mostoften carried out in fish cell lines, which simulatea natural environment for intracellular replication.During the first report of P. salmonis, the bac-terium was unable to grow on any bacteriologicalmedia tested and therefore tissue culture was usedinstead (Fryer et al. 1990). Growth has since beenachieved in several agar and broth formulationsenabling rapid growth and identification to occur.

Bacteriological media eliminate the constraints ofadditional cell purification for genetic and serolog-ical studies (Yanez et al. 2013). It could also aidin comparative analyses of antigenic changes invarious bacteriological media. Serial passages ofP. salmonis in liquid and agar culture do notappear to alter growth kinetics and virulence, butdue to a lack of in vivo lethal dose studies, moreinformation could be uncovered. Additional opti-mization of published media could furtherincrease the ability to culture P. salmonis at higherbacterial densities, provide additional insight onthe bacterium’s nutritional requirements andimprove the overall ease of in vitro culture.

Acknowledgements

The authors thank Dr John Singer for editorial com-ments. This work is based upon research supportedin part by Hatch Grant #ME0-H1-00513-13 fromthe USDA National Institute of Food and Agricul-ture. Maine Agriculture and Forestry ExperimentalStation (MAFES) #3498.

References

Almendras F.E. & Fuentealba I.C. (1997) Salmonid rickettsial

septicemia caused by Piscirickettsia salmonis: a review.Diseases of Aquatic Organisms 29, 137–144.

Barker J., Farrell I.D. & Hutchison J.G.P. (1986) Factors

affecting growth of Legionella pneumophila in liquid media.

Journal of Medical Microbiology 22, 97–100.

Benediktsdottir E., Helgason S. & Gudmundsdottir S. (1991)

Incubation time for the cultivation of Renibacteriumsalmoninarum from Atlantic salmon, Salmo salar L.,broodfish. Journal of Fish Diseases 14, 97–102.

Birkbeck T.H., Griffen A.A., Reid H.I., Laidler L.A. &

Wadsworth S. (2004) Growth of Piscirickettsia salmonis tohigh titers in insect tissue culture cells. Infection andImmunity 72, 3693–3694.

Bravo S. & Campos M. (1989) Coho salmon syndrome in

Chile. American Fisheries Society/Fish HealthSection Newsletter 17, 3.

Corbeil S., Mccoll K. & Crane M. (2003) Development of a

TaqMan quantitative PCR assay for the identification of

Piscirickettsia salmonis. Bulletin of the European Association ofFish Pathologists 23, 95–101.

Cvitanich J.D., Garate N.O. & Smith C.E. (1991) The

isolation of a rickettsia-like organism causing disease and

mortality in Chilean salmonids and its confirmation by

Koch’s postulate. Journal of Fish Diseases 14, 121–145.

Daly J.G. & Stevenson R.M. (1985) Charcoal agar, a new

growth medium for the fish disease bacterium Renibacteriumsalmoninarum. Applied and Environment Microbiology 50,868–871.

11


� 2016


Evelyn T.P.T. (1977) An improved growth medium for the

kidney disease bacterium and some notes on using the

medium. Bulletin – Office International des �Epizooties 87,511–513.

Ewann F. & Hoffman P.S. (2006) Cysteine metabolism in

Legionella pneumophila: characterization of an L-cystine-

utilizing mutant. Applied and Environment Microbiology 72,3993–4000.

Feeley J.C., Gibson R.J., Gorman G.W., Langford N.C.,

Rasheed J.K., Mackel D.C. & Baine W.B. (1979) Charcoal-

yeast extract agar: primary isolation medium for Legionellapneumophila. Journal of Clinical Microbiology 10, 437–441.

Fryer J.L. & Hedrick R.P. (2003) Piscirickettsia salmonis: aGram-negative intracellular bacterial pathogen of fish.

Journal of Fish Diseases 26, 251–262.

Fryer J.L. & Lannan C.N. (1996) Rickettsial infections of fish.

Annual Review of Fish Diseases 6, 3–13.

Fryer J.L. & Mauel M.J. (1997) The rickettsia: an emerging

group of pathogens in fish. Emerging Infectious Diseases 3,137–144.

Fryer J.L., Lannan C.N., Garces L.H., Larenas J. & Smith

P.A. (1990) Isolation of a rickettsiales-like organism from

diseased Coho salmon (Oncorhynchus kisutch) in Chile. FishPathology 25, 107–114.

Fryer J.L., Lannan C.N., Giovannoni S.J. & Wood N.D.

(1992) Piscirickettsia salmonis gen. nov., sp. nov., thecausative agent of an epizootic disease in salmonid fishes.

International Journal of Systematic Bacteriology 42, 120–126.

Gollas-Galv�an T., Avila-Villa L.A., Mart�ınez-Porchas M. &

Hernandez-Lopez J. (2014) Rickettsia-like organisms from

cultured aquatic organisms, with emphasis on necrotizing

hepatopancreatitis bacterium affecting penaeid shrimp: an

overview on an emergent concern. Reviews in Aquaculture 6,256–269.

G�omez F., Henr�ıquez V. & Marshall S. (2009) Additional

evidence of the facultative intracellular nature of the fish

bacterial pathogen Piscirickettsia salmonis. Archivos deMedicina Veterinaria 41, 261–267.

Henriquez M., Gonzalez E., Marshall S.H., Henriquez V.,

Gomez F.A., Martinez I. & Altamirano C. (2013) A novel

liquid medium for the efficient growth of the salmonid

pathogen Piscirickettsia salmonis and optimization of culture

conditions. PLoS One 8, e71830.

Hirvel€a-Koski V. (2008) The fish pathogen Renibacteriumsalmoninarum: growth in a microaerophilic atmosphere.

Veterinary Microbiology 127, 191–195.

Jamett A., Aguayo J., Miquel A., Muller I., Arriagada R.,

Becker M.I., Valenzuela P. & Burzio L.O. (2001)

Characteristics of monoclonal antibodies against

Piscirickettsia salmonis. Journal of Fish Diseases 24, 205–215.

Lannan C.N. & Fryer J.L. (1991) Recommended methods for

inspection of fish for the salmonid rickettsia. Bulletin of theEuropean Association of Fish Pathologists 11, 135–136.

Lannan C.N., Winton J.R. & Fryer J.L. (1984) Fish cell lines:

establishment and characterization of nine cell lines from

salmonids. In Vitro 20, 671–676.

Larenas J.J., Bartholomew J., Troncoso O., Fernandez S.,

Ledezma H., Sandoval N., Vera P., Contreras J. & Smith P.

(2003) Experimental vertical transmission of Piscirickettsiasalmonis and in vitro study of attachment and mode of

entrance into the fish ovum. Diseases of Aquatic Organisms56, 25–30.

Marshall S.H. & Tobar J.A. (2014) Vaccination againstPiscirickettsiosis, in Fish Vaccination. (eds R. Gudding, A.

Lillehaug and Ø. Evensen), John Wiley & Sons, Ltd,

Chichester, UK. doi: 10.1002/9781118806913.ch21.

Marshall S., Heath S., Henriquez V. & Orrego C. (1998)

Minimally invasive detection of Piscirickettsia salmonis incultivated salmonids via the PCR. Applied and EnvironmentMicrobiology 64, 3066–3069.

Marshall S.H., Gomez F.A., Ramirez R., Nilo L. & Henriquez

V. (2012) Biofilm generation by Piscirickettsia salmonisunder growth stress conditions: a putative in vivo survival/

persistence strategy in marine environments. Research inMicrobiology 163, 557–566.

Mauel M., Giovannoni S. & Fryer J. (1996) Development of

polymerase chain reaction assays for detection, identification,

and differentiation of Piscirickettsia salmonis. Diseases ofAquatic Organisms 26, 189–195.

Mauel M.J., Ware C. & Smith P.A. (2008) Culture of

Piscirickettsia salmonis on enriched blood agar. Journal ofVeterinary Diagnostic Investigation 20, 213–214.

Mccarthy U.M., Bron J.E., Brown L., Pourahmad F., Bricknell

I.R., Thompson K.D., Adams A. & Ellis A.E. (2008)

Survival and replication of Piscirickettsia salmonis in rainbow

trout head kidney macrophages. Fish & Shellfish Immunology25, 477–484.

Mikalsen J., Skjaervik O., Wiik-Nielsen J., Wasmuth M.A. &

Colquhoun D.J. (2008) Agar culture of Piscirickettsiasalmonis, a serious pathogen of farmed salmonid and marine

fish. FEMS Microbiology Letters 278, 43–47.

Omsland A., Cockrell D.C., Fischer E.R. & Heinzen R.A.

(2008) Sustained axenic metabolic activity by the obligate

intracellular bacterium Coxiella burnetii. Journal ofBacteriology 190, 3203–3212.

Omsland A., Cockrell D.C., Howe D., Fischer E.R., Virtaneva

K., Sturdevant D.E., Porcella S.F. & Heinzen R.A. (2009)

Host cell-free growth of the Q fever bacterium Coxiellaburnetii. Proceedings of the National Academy of Sciences ofthe United States of America 106, 4430–4434.

Omsland A., Hackstadt T. & Heinzen R.A. (2013) Bringing

culture to the uncultured: Coxiella burnetii and lessons for

obligate intracellular bacterial pathogens. PLoS Pathogens 9,e1003540.

Otterlei A., Brevik Ø.J., Jensen D., Duesund H., Sommerset

I., Frost P., Mendoza J., Mckenzie P., Nylund A. &

Apablaza P. (2016) Phenotypic and genetic characterization

of Piscirickettsia salmonis from Chilean and Canadian

salmonids. BMC Veterinary Research 12, 55.

Pulgar R., Hodar C., Travisany D., Zuniga A., Dominguez C.,

Maass A., Gonzalez M. & Cambiazo V. (2015)

Transcriptional response of Atlantic salmon families to

Piscirickettsia salmonis infection highlights the relevance of the

iron-deprivation defence system. BMC Genomics 16, 495.

12


� 2016


http://dx.doi.org/10.1002/9781118806913.ch21

Ramirez R., Gomez F.A. & Marshall S.H. (2015) The

infection process of Piscirickettsia salmonis in fish

macrophages is dependent upon interaction with host-cell

clathrin and actin. FEMS Microbiology Letters 362, 1–8.

Rojas V., Galanti N., Bols N.C., Jimenez V., Paredes R. &

Marshall S.H. (2010) Piscirickettsia salmonis inducesapoptosis in macrophages and monocyte-like cells from

rainbow trout. Journal of Cellular Biochemistry 110, 468–476.

Rozas M. & Enriquez R. (2014) Piscirickettsiosis and

Piscirickettsia salmonis in fish: a review. Journal of FishDiseases 37, 163–188.

Skarmeta A.M., Henriquez V., Zahr M., Orrego C. &

Marshall S.H. (2000) Isolation of a virulent Piscirickettsiasalmonis from the brain of naturally infected coho salmon.

Bulletin of the European Association of Fish Pathologists 20,261–264.

Smith P.A., Reveco F., Contreras J., Rojas M.E., Venegas C.A.

& Guajardo A. (2010) Infectivity study of Piscirickettsiasalmonis in CHSE-214 cells by confocal and transmission

electron microscopy. Bulletin of the European Association ofFish Pathologists 30, 128.

Smith P.A., Diaz F.E., Rojas M.E., Diaz S., Galleguillos M. &

Carbonero A. (2015) Effect of Piscirickettsia salmonisinoculation on the ASK continuous cell line. Journal of FishDiseases 38, 321–324.

Tobar J.A., Jerez S., Caruffo M., Bravo C., Contreras F.,

Bucarey S.A. & Harel M. (2011) Oral vaccination of

Atlantic salmon (Salmo salar) against salmonid rickettsial

septicaemia. Vaccine 29, 2336–2340.

Vera T., Isla A., Cuevas A. & Figueroa J. (2012) A new liquid

culture medium for the pathogen Piscirickettsia salmonis.Archivos de Medicina Veterinaria 44, 273–277.

Wilhelm V., Miquel A., Burzio L.O., Rosemblatt M., Engel

E., Valenzuela S., Parada G. & Valenzuela P.D. (2006) A

vaccine against the salmonid pathogen Piscirickettsia salmonisbased on recombinant proteins. Vaccine 24, 5083–5091.

Yanez A.J., Valenzuela K., Silva H., Retamales J., Romero A.,

Enriquez R., Figueroa J., Claude A., Gonzalez J., Avendano-

Herrera R. & Carcamo J.G. (2012) Broth medium for the

successful culture of the fish pathogen Piscirickettsia salmonis.Diseases of Aquatic Organisms 97, 197–205.

Yanez A.J., Silva H., Valenzuela K., Pontigo J.P., Godoy M.,

Troncoso J., Romero A., Figueroa J., Carcamo J.G. &

Avendano-Herrera R. (2013) Two novel blood-free solid

media for the culture of the salmonid pathogen Piscirickettsiasalmonis. Journal of Fish Diseases 36, 587–591.

Yanez A.J., Valenzuela K., Matzner C., Olavarria V., Figueroa

J., Avendano-Herrera R. & Carcamo J.G. (2014) Broth

microdilution protocol for minimum inhibitory

concentration (MIC) determinations of the intracellular

salmonid pathogen Piscirickettsia salmonis to florfenicol and

oxytetracycline. Journal of Fish Diseases 37, 505–509.

Yuksel S.A., Thompson K.D., Ellis A.E. & Adams A. (2006)

Improved purification of Piscirickettsia salmonis using Percoll

gradients. Journal of Microbiol Methods 66, 251–262.

Received: 22 August 2016Revision received: 4 October 2016Accepted: 5 October 2016

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