DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 98: 249254, 2012doi: 10.3354/dao02441

Published April 26

INTRODUCTION

The blue-striped snapper Lutjanus kasmira (Perci-formes, Lutjanidae), or taape, is a widespread tropi-cal marine fish. It was introduced in the 1950s intoOahu, Hawaii, USA, from Tahiti and the Marquesasas a food and sport fish and has since spreadthroughout the Hawaiian archipelago (Randall 1987).Studies of the microparasites of this fish revealed ahigh prevalence of splenic coccidia (47%) and smallintracellular Gram-negative bacteria (22%) in thekidney, which, based on light microscopy, were com-patible in morphology with epitheliocysts (Work et al.2003). While the coccidia did not seem to cause sig-nificant pathological changes, intracellular bacteriastimulated a mononuclear inflammatory responseand increased in prevalence with the size of the fishbut were absent in the largest size classes. Given

their similarity to epitheliocysts based on lightmicroscopy, these organisms were provisionallynamed epitheliocystis-like organisms (ELO) (Work etal. 2003). However, epitheliocystis in fish typicallyaffects the gill (Nowak & LaPatra 2006), whereas inHawaii the organisms were in the kidney, and theirmorphology seen under electron microscopy was notcompatible with epitheliocysts, as described by oth-ers (Wolke et al. 1970, Groff et al. 1996).

Recently, molecular approaches have confirmedthat Chlamydiae are the agents in epitheliocysts andvarious lineages are involved (Draghi et al. 2004,2007, Meijer et al. 2006, Karlsen et al. 2008). In thisstudy, we used polymerase chain reactions (PCR) andsequencing of 16S rDNA to show that these Gram-negative bacteria-like organisms (BLO) belong toChlamydiae. This represents the first reported caseof chlamydial infection in fish affecting the kidneys.

Inter-Research 2012 www.int-res.com*Email: corsaro@voila.fr

NOTE

Candidatus Renichlamydia lutjani, a Gram-negative bacterium in internal organs of

blue-striped snapper Lutjanus kasmira from Hawaii

Daniele Corsaro1,2,*, Thierry M. Work3

1CHLAREAS Chlamydia Research Association, 12 rue du Maconnais, 54500 Vandoeuvre-ls-Nancy, France2Laboratory of Soil Biology, Institute of Biology, University of Neuchtel, 2000 Neuchtel, Switzerland

3Honolulu Field Station, National Wildlife Health Center, US Geological Survey, PO Box 50167, Honolulu, Hawaii 96850, USA

ABSTRACT: The blue-striped snapper Lutjanus kasmira (Perciformes, Lutjanidae) are cosmopoli-tan in the Indo-Pacific but were introduced into Oahu, Hawaii, USA, in the 1950s and have sincecolonized most of the archipelago. Studies of microparasites in blue-striped snappers from Hawaiirevealed chlamydia-like organisms (CLO) infecting the spleen and kidney, characterized by intra-cellular basophilic granular inclusions containing Gram-negative and Gimenez-positive bacteriasimilar in appearance to epitheliocysts when seen under light microscopy. We provide molecularevidence that CLO are a new member of Chlamydiae, i.e. Candidatus Renichlamydia lutjani, thatrepresents the first reported case of chlamydial infection in organs other than the gill in fishes.

KEY WORDS: Epitheliocystis Chlamydia-like organism Chlamydiae Blue-striped snapper Lutjanus kasmira

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Dis Aquat Org 98: 249254, 2012

MATERIALS AND METHODS

Fish were collected from Oahu, Hawaii, as part ofgeneral surveillance for microparasites in reef fish(Work et al. 2003, 2010). For light microscopy, caudalkidneys were fixed in 10% neutral buffered formalin,embedded in paraffin, sectioned at 5 m and stainedwith hematoxylin and eosin, Gram (Prophet et al.1992) or Gimenez (Gimenez 1964). The latter stain isroutinely used to confirm chlamydia using lightmicroscopy (Nabeya et al. 1991). Paired sampleswere preserved in 95% ethanol or DMSO/EDTA/NaCl (Dawson et al. 1998) for DNA extraction. Sam-ples were confirmed as BLO-positive or BLO-nega-tive based on examination of histology slides. DNAwas extracted from kidneys of affected (n = 3) andunaffected (n = 3) fishes using a Wizard SV GenomicDNA (Promega) kit following the manufacturersinstructions.

PCR assays were conducted with pan-chlamydia16S rDNA primers CF1 (5-CGT GGA TGA GGCATG CRA GTC G-3), CR6 (5- GTC ATC RGC CYY-ACC TTV SRC RYY TCT-3) and CR7 (5-TAC CTTGTT ACG ACT TMA YCC YAG-3) as described byCorsaro et al. (2002), under reaction conditions of aninitial 5 min at 95C, followed by 40 cycles of 1 min at94C, 30 s at 60 or 62C and 1 min 30 s at 72C, and afinal extension of 10 min at 72C. Purified PCR prod-ucts were sequenced with a set of inner primers, andthe obtained sequences were compared with Gen-Bank database sequences using the basic local align-ment search tool (BLAST) (Altschul et al. 1990). Amaster alignment, including closest relatives andrepresentatives of the major chlamydial lineages,was produced using ClustalW (Larkin et al. 2007),and phylogenetic analyses were performed by apply-ing analyses for distance (neighbor joining, NJ) andmaximum parsimony (MP) with MEGA5 (Tamura etal. 2011) and maximum likelihood (ML, GTR+I+model) with TREEFINDER (Jobb et al. 2004), using1000 bootstrap replicates. Similarity sequence valueswere calculated using BioEdit (Hall 1999).

The nearly full-length 16S rDNA sequence of theBLO infecting the blue-striped snapper Lutjanus kas-mira is available in GenBank under the accession no.JN167597.

RESULTS

Fish affected with BLO showed no evident grosslesions or adverse clinical signs. Based on micro -scopy, the organisms consisted of variably sized cir-

cular clusters of Gram-negative bacteria. In putativeearly infections, bacteria were surrounded by lym-phocytes, whereas in putative chronic infections,bacteria were surrounded by a capsule consisting ofcollagen mixed with plump fibroblasts. The bacteriastained positive with Gimenez stain and were Gram-negative (Fig. 1).

We initially obtained a 300 base pair (bp) fragmentfrom an ethanol-preserved sample, which showed85% sequence identity with the uncultured rhab-dochlamydia CN808 (GenBank accession no.EU090709), recovered from a human respiratorysample (Haider et al. 2008). PCR from unaffected fishsamples were negative or gave non-specific se -quences. Nearly full 16S rDNA sequences were sub-sequently obtained from DMSO/EDTA/NaCl-pre-served kidneys originating from 3 infected fish. In apair-wise sequence analysis without indels, BLOwere 91.7% identical with the same phylotypeCN808 and 88.2 to 88.7% identical with Rhab-dochlamydia spp. Similarity values of the BLO andCN808 with other Rhabdochlamydiaceae rangedfrom 88.0 to 90.3%, whereas within the remainingRhabdochlamydiaceae, values ranged from 88.0 to94.4%. The similarities of BLO were 85 to 86.7% withthe sister-group of Simkaniaceae and

Corsaro & Work: Identification of epitheliocystis-like organism

phology from the 2 Rhabdochlamydia spp. recoveredfrom arthropods, which have elongate rickettsiella-like 5-layered elementary bodies (Kostanjsek et al.2004, Corsaro et al. 2007). Similarly, based on elec-tron microscopy (Work et al. 2003), BLO differ fromepitheliocystis agents infecting gills of fish (Wolke etal. 1970). BLO and Rhabdochlamydia are relativelydistant genetically, and the closest relative of BLO isthe uncultured strain CN808 of unknown morphol-ogy recovered from a human respiratory sample

(Haider et al. 2008). Moreover, Rhabdochlamydi-aceae as well as Simkaniaceae show a large diversityin putative genus-level phylotypes (i.e. 16S rDNAsimilarities

Dis Aquat Org 98: 249254, 2012

tence of chlamydia-like morphologies in Simkani-aceae and paraphyletically in some Rhab doch lamy -dia ceae, with the development of rickettsiella-likeelementary bodies only in the sublineage infectingarthropods.

Since the first description of epitheliocystis byHoffman et al. (1969), chlamydiae were suggested tobe the causative agents based on morphology. Thiswas recently confirmed by molecular approaches tar-geting the 16S rDNA. Nearly full sequences revealed

2 novel Chlamydiae affecting salmonids, CandidatusPiscichlamydia salmonis and Candidatus Clavo -chlamydia (= Clavichlamydia) salmonicola (Draghi etal. 2004, 2010, Karlsen et al. 2008), whereas partialsequences revealed the involvement of Neochlamy-dia sp. (Parachlamydiaceae) and other phylotypes(Meijer et al. 2006, Draghi et al. 2007, 2010, Polking-horne et al. 2010) falling within Rhabdochlamydi-aceae and the E6 lineage (Corsaro & Venditti 2009).Simkaniaceae also might be epitheliocystis agents,

252

0.03

Piscichlamydia salmonis (AY462244)

IPI_772-868-964

IPI_862-958-1054

Ca. Renichlamydia lutjani

IPI-884-980

IPI_796-892IPI_831-927-1023

92/82/30

89/99/98

85/85/52

100/92/97

99/99/93

97/99/96

75/55/-

81/81/35

97/71/45

100/99/93

74/-/-

90/87/69

89/98/60

97/94/92

73/69/39

62/-/52

65/31/20

89/95/79

99/100/9361/72/73

71/66/-

88/95/-

64/-/-

88/100/96

68/-/27

70/64/49

100/90/88

92/100/80

98/100/9986/69/-

94/48/-

92/86/92

90/91/82

54/41/-

82/30/-

95/82/65

86/88/-

52/66/-

70/-/- Parachlamydia acanthamoebae (AM941720)

cvE70 (freshwater pond, JF706725)

LD1-PA25

Protochlamydia naegleriophila (DQ632609)

Estrella lausannensis (WTP, Spain, EU074225)

CK-2C2-23 (bivalve sediment, Florida, EU488135)

Protochlamydia amoebophila (AF083615)

Neochlamydia sp. UWC22 (AF083616)

p22m06ok (FJ479189)

cvE71 (freshwater pond, JF706724)

cvE65 (JF706723)

cvE60 (freshwater pond, France, FJ976107)

Waddlia malaysiensis (AY184804)

(anoxic marine sediment, Scotland, AY114316, AY114327)

Waddlia chondrophila (AF042496)

Criblamydia sequanensis (WTP, France, DQ124300)

Neochlamydia hartmannellae (AF177275)

Metachlamydia lacustris (GQ221847)MABRDTU43 (nitrifying bioreactor, FJ529996)

UWE1 (AF083614)

LD1-PA42

PRPR10 (poriferan, Hawaii, DQ903988)

LT100PlH9 (Lake Tanganyika, DQ444409)

GP27685gO2 (yellow microbial mat, Azores, HM445488)

cvE99 (JF513057)

Simkania negevensis (U68460)

CRIB34 (WTP, Spain, EU363465)

EP912A0051 (white microbial mat, Hawaii, HM444986)

Xenoturbella endosymbiont (EF177461)

EPR4059-B2-Bc66 (seafloor lava, EU491566)

CN808 (human respiratory sample, EU090709)

Fritschea bemisiae (AY140910)

EP912A0005 (white microbial mat, Hawaii, HM444977)

PRPR85 (poriferan, Hawaii, DQ903997)

Rhabdochlamydia crassificans (AY928092)

CN554 (human respiratory sample, EU090707)

cvE55 (freshwater pond, France, FJ976100)

FFCH17845 (grass prairie, EU133918)

cvE6 (freshwater pond, France, AF448722)

LT110PlE2 (Lake Tanganyika, DQ444442)

Rhabdochlamydia porcellionis (AY223862)

CRIB33 (WTP, Spain, EU683887)

E9 clade (freshwater ponds, AF448723, FJ976095)

Amazon clade (AY326517, AY326519, FJ976093)

cvE18 (freshwater pond, Italy, FJ976098)

Chlamydophila pneumoniae TW183 (L06108)

P9X2b3G08 (seafloor lava, EU491150)

Chlamydophila psittaci NJ1 (U68419)Chlamydia trachomatis L2 (U68443)

Clavichlamydia salmonicola (DQ011662)clone122 (fecal sample from sea birds, GU068510)

CRIB32 (WTP, Spain, EU363464)

Rhabdochlamydiaceae

Simkaniaceae

E6 lineage

E60 lineage

E71 lineage

Parachlamydiaceae

Criblamydiaceae

Chlamydiaceaeand relatives

Waddliaceae

Piscichlamydiaceae

98/99/97

(coastal lake, Italy)

98/93/82

cvE88 (JF513056)

Fig. 2. Maximum-likelihood phylogenetic tree of the Chlamydiae. The novel chlamydia Candidatus Renichlamydia lutjani re-trieved in this study is in bold. Origins and GenBank accession nos. are in parentheses. Numbers at the nodes represent boot-strap values obtained by maximum likelihood (ML)/neighbor joining (NJ)/ maximum parsimony (MP) after 1000 replications;

filled circles represent values of 100% obtained with all 3 methods. WTP: water treatment plant

Corsaro & Work: Identification of epitheliocystis-like organism

with the undescribed salmon symbiont from gills(GenBank acc. no. EU326493).

Epitheliocystis is an infectious disease affectingthe gills of several marine and freshwater teleostfishes (Corsaro & Venditti 2004, Nowak & LaPatra2006), although infections have been occasionallyreported in chondrichthyan fishes (Borucinska &Frasca 2002, Polkinghorne et al. 2010). Infectionwith epitheliocystis is limited to the gills and lesscommonly the skin and oral cavity. Infections havenever been documented in internal organs. In con-trast, other pleomorphic intracellular pathogens, i.e.Piscirickettsia, Francisella (Gammaproteobacteria)(Mauel & Miller 2002, Mauel et al. 2003, Mauel etal. 2005) and at least a case of Neorickettsia (Alpha -pro teo bacteria) (Pusterla et al. 2000), cause systemicinfections in fish affecting internal organs like thekidney, liver and spleen. No cyst-like enlarged cellsare formed. Our findings thus identify anotherorganism belonging to Chlamydiae that infects theinternal organs of fish. Demographic studies suggestthat this parasite is directly transmitted and may beof limited pathogenicity (Work et al. 2010). Futurestudies should include solidifying these findings byother methods, such as in situ hybridization or im -muno histochemistry.

Systematic description

Name: Candidatus Renichlamydia lutjani. Etymology: Candidatus, category for obligate in -

tracellular bacteria. Renichlamydia n. gen. Reni, L.n.renes, the kidneys; Chlamydia, N.L. fem. n., a bacte-rial genus name; Renichlamydia N.L. fem. n., kidneychlamydia. Ca. Renichlamydia lutjiani n. sp., lutjianiN.L. gen. sing. n., of Lutjanus, genus name of the fishhost.

Physical description: Spherical cells 1 to 2 m indiameter, with crystalloid or electron-dense gran-ules, inside a thin inclusion membrane, surroundedby a granular matrix with mitochondria and an exter-nal thick capsule of collagen and fibroblasts (Work etal. 2003).

Pathology: Fish are infected mainly in the kidney,occasionally in the spleen.

Natural host: Blue-striped snapper Lutjanus kas-mira.

Distribution: Pacific Islands (Work et al. 2010).

Acknowledgements. D.C. was partially supported by theNovartis Foundation. Mention of products or trade namesdoes not imply endorsement by the USA Government.

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Editorial responsibility: Dieter Steinhagen,Hannover, Germany

Submitted: June 28, 2011; Accepted: January 10, 2012Proofs received from author(s): March 26, 2012

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