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Short Communication *Corresponding author Xinzhong Wu, South China Sea Institute of Oceanography, Chinese Academy of Sciences, China, Identification of Rickettsia-Like Email: Submitted: 19 January 2016 Organism (RLO) in the Oyster Accepted: 17 April 2017 Published: 19 April 2017 ISSN: 2475-9430 Crassostrea rivularis Gould Copyright Yang Zhang1#, Jing Fang2#, Jingfeng Sun3, and Xinzhong Wu1,2* © 2017 Wu et al. 1South China Sea Institute of Oceanography, Chinese Academy of Sciences, China OPEN ACCESS 2Ocean College, Qinzhou University, China 3Aquaculture College, Tianjin Agriculture University, China Keywords #Yang Zhang and Jing Fang contributed equally to this work • Oyster Crassostrea rivularis Gould (also known as Crassostrea ariakensis) Abstract • Rickettsia-like organism (RLO) • 16S rDNA The oyster Crassostrea rivularis Gould (also known as Crassostrea ariakensisis) an • Phylogeny important bivalve cultured in southeastern China. Since 1992, these oysters • In situ hybridization have suffered high mortality during winter and spring. An intracellular rickettsia-like organism (RLOs) was proposed as the causative agent. In this study, RLOs were purified from the gill and digestive gland of dying oyster (C. rivularis Gould), and their 16S rDNA was amplified from the purified products. To eliminate non-specific bacterial 16S rDNA contamination, the cloned products of bacterial 16S rDNA from gill RLO were screened by the probes of bacterial 16S rDNA amplified from the digestive gland RLO. Finally, the five strongest hybridized dots were picked out and sequenced. The RLO’s 16S rDNA sequence was reconfirmed and the pathogen was found only in epithelia cells by in situ hybridization (ISH) using specific probes. Sequence alignment and phylogenetic analysis indicated the RLO bacterium found in oyster C. rivularis Gould was most similar to Piscirickettsia salmonis, and might be classified into the family of gamma .

ABBREVIATIONS infected with RLO, causing mortality and dramatic economic losses, such as the scallop, Pecten maximus [22]; the oyster RLP: Rickettsia-Like Prokaryote; RLOs: Rickettsia-Like Crassostrea virginia and the hard clam M. mercenaria Organisms; ISH: Hybridization; PBS: Phosphate-Buffered in situ pearl oyster Pinctada fucata and Pinctada maximum [18,24]; the Saline; min: minutes; h: hours; TEM: Transmission Electron oyster C. rivularis [1]; the abalone Haliotis rufescens [23]; the Microscopy; s: seconds; PCR: Polymerase Chain Reaction; SSPE: scallop Chlamys farreri (unpublished results). Although RLOs have Saline Sodium Phosphate Ethylenediaminetetraacetic Acid; AP: been recognized as an important pathogen to aquatic [25]; organisms, and the Alkaline Phosphatase; DIG: Digoxin studies have been carried out mostly on a morphological and INTRODUCTION molecular level. The oyster, Crassostrea rivularis Gould, also known as pathological level, while few studies have identified them on the Crassostrea ariakensis, is one of the most economically important cultured species in southeastern China, especially in the Guangxi, C. ariakensis, In this paper, the RLO was purified from the infected oyster Guangdong and Fujian provinces. With the large expansion of this bacterium might be classified as a member culturing, mass mortalities have occurred persistently and caused is distantly related to Piscirickettsia salmonis by phylogenetic of gamma-proteobacteria by using 16S rDNA analysis and it great economic loss since 1992 in the Guangdong province of analysis. In situ-hybridization suggested that the pathogen is China. Recent studies suggested oyster culture suffered from localized in oyster epithelia cells. severe mortality caused by the pathogen Rickettsia-like organism MATERIALS AND METHODS (RLO) [1,2]. Sample and processing Rickettsias are Gram-negative , generally described as obligate intracellular pathogens, that have been reported in The oyster, C. rivularis from Hailing Bay in Yang Xi county, GuangDong province, China, Mercenaria mercenaria, Gould, 2-3 years old, were collected 1977various [21], fishes, many crustaceans mollusk [3-15], species and have mollusks been [1,16-20]. reported toSince be dying oysters were picked out, the bodies cross-sectioned the first report by Harshbarger et al. in in October 2004 when oyster deaths occurred in the field. The

Cite this article: Zhang Y, Fang J, Sun J, Wu X (2017) Identification of Rickettsia-Like Organism (RLO) in the Oyster Crassostrea rivularis Gould. Ann Clin Cytol Pathol 3(2): 1056. Wu et al. (2017) Email:

Central Bringing Excellence in Open Access Eliminating unwanted bacterial contamination paraformaldehyde for pathogenic observation. The residual body into 5mm thick piece just above the ventricle, and fixed in 4% To eliminate unwanted bacterial 16S contamination, the 16S wasRLO stored purification at -80 until used for RLO purification. fragmentsfragment amplified from the fromgill RLO the wereoyster screened gill was byscreened probes usingfrom the Infected gills and digestive glands were used for RLO digestiveprobes from glands. the Thefragments probes of were digestive labeled glands. with biotinSixty-six according of 16S purification using renografin density gradient centrifugation Detection starter kit (Roche). The result was recorded by X-ray infected tissues were homogenized in phosphate-buffered saline to the instructions provided in DIG HIGH prime DNA labeling and [26,27] as described previously with some modifications. Briefly, 2HPO4 2PO4 filmMolecular (Koda). phylogenetic analysis (PBS,the fat, pH7.4: then, Nathe pellets, 53.9mM; were re-suspended KH , 12.8mM; in PBS, NaCl, centrifuged 72.6mM) and centrifuged at 11000×g for 40 minutes (min) at 4 to remove The nucleotide sequences of the RLO 16S rDNA DQ123914.1 at 700×g for 20 min at 4 and 1100×g for 10 min to remove cell and DQ118733.1 have been blasted within the RDP_ debris. Subsequently, supernatant fluids were collected and re- for density gradient centrifugation after being re-homogenized. ThenSeqDescByOTU_tax_outline.txt. those sequences were further A total selected of 287 by sequences OTU number, from centrifuged at 11000×g for 40 min. The pellets were then used separate infected oysters with identity >=90% were selected.

About 300mg of the re-homogenized pellets were laid on the top only those with OTU number >= 3 and with the best score were layer of discontinuous renografin gradients (15%, 20%, 25%,g selected. The phylogenetic tree was made by MEGA 3.1 [29], with 30%, 35% v/v from top to bottom in turn, each layer with about theIn situ Neighbor-Joining hybridization (NJ) Method. 5.5ml volume and 1.5 cm in depth) and centrifuged at 90,000× for 2 hours (h) at 4 (Sorvall S80). The particles concentrated at g for paraformaldehyde in PBS (pH 7.4), dehydrated in an ascending the density interfaces of 20%-25% and 25%-30% were collected, Infected tissues were dissected and fixed with 4% anddiluted stained with 5 withvolumes Uranyl of PBS, Acetate and re-centrifuged and observed at 15000× using JEOL transmission40 min. Finally, electron the pellets microscopes were diluted (TEM). to a suitable suspension ethanol series (50%, 70%, 80%, 90%, 95%, 100% v/v) for two times, followed by three washes in xylene, embedded in paraffin, RLO DNA extraction and 16S rDNA PCR amplification hydratedsectioned inat a5μm, reverse mounted ethanol on series. APES-coated The rehydrated slides, and slides baked used at for55 forin situ 4 h. Then the section was deparaffinized in -1xylene and re-

About 80mg granules purified from gills were used in DNA hybridization were digested in 30 μg ml Proteinase K extraction, as well as granules purified from digestive glands. solution (pH 8.0) under 37 for about 20 min before hybridization. DNA was extracted according to the methods of Kellner-Cousin Slides were then neutralized in 2× saline sodium phosphate-1 [28]. Briefly, purified RLOs were resuspended in TE buffer (Tris- ethylenediaminetetraacetic acid (2×SSPE, pH 7.4) buffer for HCl 10 mM, EDTA 1mM, pH 8.0) and incubated for 20 min at 10 min, and treated with prehybridization solution (0.5mg ml 37 with lysozyme (1mg/ml). Then, sodium dodecyl sulfate and salmon sperm DNA, 5×Denhardt’s reagent, and 2×SSPE) in a proteinase K were added to a final concentration of 0.5% and weremoist separately chamber atadded 42 foronto 30 two min. different After theslides prehybridization, and hybridized 100μg/ml respectively and the suspension was incubated at 55 100ng of the positive probes and the negative control (NC) probes for 3 h. Samples were extracted with phenol-chloroform (twice) at 42 for about 16 h in the moist chamber. After hybridization, and chloroform (once). Nucleic acids were precipitated with dissolved in sterile distilled water. The universal bacterial PCR unbound probes were washed off with 2×SSPE, 1×SSPE, and 100% ethanol, washed with 70% ethanol (twice), air-dried and (Alkaline phosphatase) anti-digoxin (DIG) antibody and detected 0.5×SSPE at 42. Finally, the slides were added into AP-conjugated primers were derived from the highly conserved bacterial 16S (Escherichia coli by NBT/BCIP indication reagent. region. The forward primer sequence isE. 5’-gcttaacacatgcaagtcg-3’ coli 16S rDNA positions 39-57), the reverse primer The specificity of assumed probe sequences chosen from highly variable regions of the RLO 16S rDNA sequence (GenBank sequence is 5’- actaccgattccgacttca-3’ ( 16S rDNA positions2+ sequence within the databases DDBJ-EMBL-GenBank using the 1322-1344). PCR was performed with 25μl reaction mixtures BLASTnaccession service. number The DQ123914) probe was were monolabeled confirmed with by retrieving DIG and the containing 1μl template DNA, 2.5μl 10×PCR buffer within Mg (TaKaRa, Dalian, China), 1μl of 10mM each dNTPs, 0.8U Taq polymerase (TaKaRa), and 0.5μl each of 25mM universal bacterial sequence is DIG-5’-aggtagtctgtgaataatgggctactg-3’ at the position 16S rDNA primers. The mixture was denatured at 94 for 2 min from 402-427 in DQ123914. A NC probe was also implemented before amplification. The amplification profile consisted of 30 mismatchto monitor tothe the experimental RLO sequence, conditions. but less The than NC probe 12 nucleotides sequence seconds (s) at 94, 30 s at 56 and 90 s at 72 cycled 30 times, with was DIG-5’-gggatgtaggttaataccttgcatctt-3’ with a 12 nucleotides an additional 5 min at 72 following the final cycle using Thermal database. mismatch to other bacterial 16S rDNA sequences by NCBI BLAST PX2 PCR amplifier (Thermal Ltd.). The PCR products were Theory/calculation collecteddetermined from using the 1.5% agarose agarose gel and gel cloned electrophoresis into a PMD-18T and ethidium vector In this study, it is reported that the RLO bacterium found (Takarabromide Inc.). staining. Expected PCR products (size ~1300bp) were

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Central Bringing Excellence in Open Access in oyster Crassostrea rivularis Gould was most similar to Piscirickettsia salmonis gamma proteobacteria but it necessary to develop more studies. It can provide a theoreticaland could basis be for classified analysis intoof the a new death family of the of oyster C. rivularis Gould and preventing or controlling the RLO. RESULTS RLO purification

The purified RLOs mainly existed at band 20%-25% and displayed25%-30%, particles which were containing coincident not withonly the reportRLOs, butby Lialso and some Wu cell(Li and debris Wu, and 2004). contaminated Under TEM bacteria observation, (Figure the 1). purified products Figure 1 Electron micrograph of RLOs (arrows) from the oyster gill Eliminating bacterial contamination and molecular epithelial cell, with 2% uranyl acetate. phylogenetic analysis In density gradient centrifugation, only granules with the same density should be concentrated in the same layer, while particles outwith anddiffering sequenced densities (Figure should 2). be After eliminated. being retrievedAmong the from 66 dots the GenBankin cross hybrid,database, the the 5 mostsequences strongly were reactive found to dots belong were to picked three known bacteria (Vibrio ordalii, Pseudomonas putida, Serratia marcescens) and one unknown bacterium. By comparing the morphological characters of the three known bacteria described in Bergey’s manual of systematic bacteriology (second edition, assumed2004) with to that be theof the RLO RLO sequence. found in 48oyster, sequences the morphological (Showed in Figure 2 character could not fit well. So the unknown sequence was products. Five Sixty-six of the of strongest 16S rDNA hybridized amplified dots fragment (indicated inserted by arrows) were weredetected picked by out probes and sequenced. synthesized from digestive gland purified alignmentTable 1) were analysis, finally the selected sequences and which used inare alignment similar to with the RLO two RLO sequences using the clustalX 1.83 software. By sequence be inferred that the RLO is a type of gamma proteobactium. By cagccgcggt 16S sequence all belong to the Gamma proteobacteria. Thus it can 481 aatacggagg gtccgagcgt taatcggaat tactgggcgt aaagggtgcg sequence of Piscirickettisia salmonis taggcggata wasphylogenetic not high analysis, enough the to sequence classify these was most two bacteriasimilar to into the 16S one (Figure 3), but the similarity obtained as follows: actgcataac family. In this study, 1304 sequences of 16S rDNA of RLO were 541 tgtaagtggg tagtgaaaga cctgggctca acctgggagg tgctatccaa 1 gcttaacaca tgcaagtcga gcggtaacag gaagagcttg ctctttgctg acgagcggcg agatatagga 601 tagagtacag aagaggagtg tggaatttcc tgtgtagcgg tgaaatgcgt

agaaatccgg tacgaaagcg 61 gacgggtgag taacgcgtag gaatctgact gtaagagggg gatagcccgg 661 aggaacaccg gtggcgaagg cggcactctg gtctgatact gacgctgagg 121 attaataccg cataacacct aagggtaaaa agaggcactt gtgctactgc 721 tggggagcaa acaggattag ataccctggt agtccacgct gtaaacgctg ttacagagga tctactagtc 181 gcctgcgttg gattagctag ttggtggggt aaaggcttac caaggcgacg 781 gttgggaact taaaagtttt tagtggcgaa gcaaacgcgc taagtagacc atccatagct gcctggggag 241 gctctgagag gatgatcagc cacactggga ctgagacacg gcccagactc 841 tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ctacgggagg ggtggagcat

gcgtgtgtga cggaatggcg 301 cagcagtggg gaatattgca caatggggga aaccctgatg cagccatgcc 901 gtggtttaat tcgacgcaac gcgaagaacc ttacctggtt ttgacatcct

gtgaataatg gtcgtcagct 361 agaaggcttt cgggttgtaa agcactttca gtggtgagga aaggtagtct 961 aagagatttg ccagtgcctt cgggagccga gtgacaggtg ctgcatggct 421 ggctactgtg acgttagcca cagaagaagg accggcaaac tccgtgccag

1021 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttatcc Ann Clin Cytol Pathol 3(2): 1056 (2017) 3/8 Wu et al. (2017) Email:

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Figure 3 Piscirickettsia Salmonis.

Phylogenetic analysis shows that the most similar sequence to RLO 16S rDNA is the 16S sequence of ttatttgcca

In situ1261 hybridization gcagtctgca actcgactgc gtgaagtcgg aatcggtagt aatc aggtggggac 1081 gcatgtaaag atgggaactc taaggagact gccggtgaca agccggagga 1141 gacgtcaagt catcatggcc cttacgacca gggctacaca cgtgctacaa comparing the hybridization signal produced using the RLO tggggcgtac The specificity of the bacterium probe was identified by The bacteria were recognized clearly when hybridized with the specific probe and non-specific probe under the same condition. agtccggatc 1201 aaagggaagc gaagcggtga cgtggagcca aacctatcaa agcgcctcgt with negative control probes (Figure 4A,4B). Meanwhile, ISH RLO specific probes, while no signals presented when hybridized Ann Clin Cytol Pathol 3(2): 1056 (2017) 4/8 Wu et al. (2017) Email:

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Table 1: Sequence name used in alignment and phylogenetic analysis. AccessionId AlignI DESC Bacteria; ProteoBacteria; DeltaproteoBacteria; Desulfobacterales; DED1 Desulfobacteraceae; Desulfobacula HMMVBeg-47 Bacteria; Proteobacteria; Deltaproteobacteria; Desulfobacterales; AJ704694.1 marine sediment clone AJ704694.1 DED2 Desulfobacteraceae; Desulfobacula Bacteria; Proteobacteria; ; Pasteurellales; AY177803.1 PAA1 AY177803.1 Antarctic sediment clone SB4_98 Pasteurellaceae; Actinobacillus Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; AY465366.1 PAA2 AY465366.1 Actinobacillus rossii str. JF2073 Pasteurellaceae; Actinobacillus Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; AY465368.1 PAP1 AY465368.1 Actinobacillus rossii str. P.. 12 Pasteurellaceae; Pasteurella Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; AF139582.1 PAP2 AF139582.1 Pasteurella aerogenes str. JF2039 Pasteurellaceae; Pasteurella AY465358.1 Pasteurella aerogenes str. 4-97; AY465358.1 Bacteria; Proteobacteria; Gammaproteobacteria; EstimateJF2420 Microbial Burden and Commercial MOA1 Pseudomonadales; Moraxellaceae; Acinetobacter AirlineEU341176.1 Cabin Evaluation Air commercial Rapid aircraft Technologies cabin air EU341176.1 Bacteria; Proteobacteria; Gammaproteobacteria; MOA2 Pseudomonadales; Moraxellaceae; Acinetobacter clone AV_4R-S-C13 Bacteria; Proteobacteria; Gammaproteobacteria; DQ834360.1 PSP1 DQ834360.1 Acinetobacter sp. str. BYC2 Pseudomonadales; Pseudomonadaceae; Pseudomonas Bacteria; Proteobacteria; Gammaproteobacteria; AY486375.1 PSP2 AY486375.1 Pseudomonas sp. str. AU2390 Pseudomonadales; Pseudomonadaceae; Pseudomonas Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Piscir AY486377.1 PIP1 AY486377.1 Pseudomonas sp. str. AU4899 ickettsiaceae;Piscirickettsia Bacteria; Proteobacteria; Gammaproteobacteria; Thiotrichales; AY498633.1 PIP2 AY498633.1 Piscirickettsia salmonis IRE-91A Piscirickettsiaceae; Piscirickettsia Bacteria; Proteobacteria; Gammaproteobacteria; AY498636.1 XAP1 AY498636.1 Piscirickettsia salmonis SCO-95A Xanthomonadales; Xanthomonadaceae; Pseudoxanthomonas Bacteria; Proteobacteria; Gammaproteobacteria; EU177791.1EU250940.1 Pseudoxanthomonas sp. str. Ca7- EU177791.1EU250940.1 XAP2 Xanthomonadales; Xanthomonadaceae; Pseudoxanthomonas NFC7-F12 Bacteria; Proteobacteria; Gammaproteobacteria; AB218877.1 XAS1 Xanthomonadales; Xanthomonadaceae; Schineria 1J03 AB218877.1 Koukoulia aurantiaca str. IAM Bacteria; Proteobacteria; Gammaproteobacteria; XAS2 response15137 lab rearing and antibiotic treatment Xanthomonadales; Xanthomonadaceae; Schineria EF608545.1 predatory Poecilus chalcites their EF608545.1 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; IDI1 Idiomarinaceae; Idiomarina digestive tract ground beetle clone PCD-40 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; DQ337031.1 subsurface water clone DQ337031.1 IDI2 Idiomarinaceae; Idiomarina EV818EB5CPSAJJ20 DQ235576.1 biofilm population water pipeline DQ235576.1 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; augmentationsbiofilms steel pipelines chromium Gulf contaminated Mexico clone wastes 100 IDP1 Idiomarinaceae; Pseudidiomarina DQ899878.1 structure receiving long-term DQ899878.1 Cr(VI) contamination clone G1DMC-174 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; landfill sediments Gorwa industrial estate IDP2 Idiomarinaceae; Pseudidiomarina DQ234155.2 determined library mangrove DQ234155.2 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; augmentationsclone DS071 chromium contaminated wastes IDU1 DQ899898.1 structure receiving long-term DQ899898.1 Bacteria;Idiomarinaceae; Proteobacteria; unclassified_Idiomarinaceae Gammaproteobacteria; Alteromonadales; landfill sediments Gorwa industrial estate IDU2 Cr(VI)str. JB11 contamination clone G2DMC-116 AY345388.1 Loihi submarine volcano isolate AY345388.1 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; Idiomarinaceae; unclassified_Idiomarinaceae

AY532642.1 INU1 AY532642.1 Bugula simplex symbiont Bacteria;Incertae; unclassified_IncertaeProteobacteria; Gammaproteobacteria; Alteromonadales; SurfaceDQ351747.1 Sediments Microbial marine Adherent sediments Sediment clone Particles Heavy Metal Contaminated North Sea DQ351747.1 INU2 Bacteria;Incertae;unclassified_Incertae Proteobacteria; Gammaproteobacteria; Alteromonadales; Belgica2005/10-120-16

EU399549.1 INM1 EU399549.1 Marinobacter sp. str. BR-13 Incertae; Marinobacter; Unclassified

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Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales;

Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; DQ015835.1 Antarctic lake water clone ELB19- DQ015835.1 INM2MOM1 Incertae;Moritellaceae; Marinobacter; Moritella Unclassified 223 Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; AY394860.1 MOM2 AY394860.1 Moritella sp. str. 762 G Moritellaceae; Moritella Bacteria; Proteobacteria; Gammaproteobacteria; Alteromonadales; AY380781.1 Moritella viscosa str. AY380781.1 SHS1 Shewanellaceae; Shewanella 2002/09/1069-1 Bacteria;Proteobacteria; Gammaproteobacteria; Alteromonadales; AB003190.1 SHS2 AB003190.1 Shewanella sp. str. SC2A Shewanellaceae; Shewanella Bacteria; Proteobacteria; Gammaproteobacteria; Chromatiales; AF132875.1 Shewanella frigidimarina str. AF132875.1 CHR1 ; Rheinheimera ACAM 533 Bacteria; Proteobacteria; Gammaproteobacteria; Chromatiales; AY241547.1 aggregates water column German AY241547.1 CHR2 Chromatiaceae; Rheinheimera Wadden Sea part North Sea isolate str. HP1 HP1 AJ441080.1 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; AJ441080.1 Rheinheimera baltica str. OSBAC1

AY136145.1 ENU1 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; AY136145.1 Cacopsylla pyri symbiont Enterobacteriaceae; unclassified_Enterobacteriaceae symbiont AY136162.1 Uroleucon nigrotuberculatum AY136162.1 ENU2 Bacteria;Enterobacteriaceae; Proteobacteria; unclassified_Enterobacteriaceae Gammaproteobacteria; Legionellales; abundant members communty tallgrass soil LEL1 Legionellaceae; Legionella undisturbedEU134750.1 mixedevolutionary grass prairie between preserve rare and clone EU134750.1 Bacteria; Proteobacteria; Gammaproteobacteria; Legionellales; LEL2 Legionellaceae; Legionella FFCH14647 Bacteria; Proteobacteria; Gammaproteobacteria; Legionellales; EU250248.1 LEU1 EU250248.1 acid mine drainage clone GXDC-34

AY536230.1 AY536230.1 host gut clone LAgut--P18 Bacteria;Legionellaceae; Proteobacteria; unclassified_Legionellaceae Gammaproteobacteria; Legionellales; abundant members communty tallgrass soil LEU2 undisturbedEU134792.1 mixedevolutionary grass prairie between preserve rare and clone EU134792.1 Legionellaceae; unclassified_Legionellaceae Bacteria; Proteobacteria; Gammaproteobacteria; ; OCU1 oneFFCH4066 cell time Boothbay Harbor 1m depth clone EF202341.1 Matching and function marine EF202341.1 ;Bacteria; Proteobacteria; unclassified_Oceanospirillaceae Gammaproteobacteria; Oceanospirillales; OCU2 MS024-3A

EF516584.1 Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; EF516584.1 grassland soil clone FCPP727 Oceanospirillaceae; unclassified_Oceanospirillaceae

AY922202.1 OCN1 Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; AY922202.1 whalefall clone 131636 Oceanospirillaceae; Nitrincola Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; AY567473.1 SAS1OCN2 AY567473.1 Nitrumincola lacisaponis str. 4CA Saccharospirillaceae;Oceanospirillaceae; Nitrincola Saccharospirillum Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; AJ315984.1 SAS2 AJ315984.1 Arhodomonas sp. str. EL-201 Saccharospirillaceae; Saccharospirillum AJ315983.1 Saccharospirillum impatiens str. AJ315983.1 Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; EL-105 = DSM 12546 Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; DQ123914.1 OCO1DQ12 DQ123914.1 Oceanrickettsia ariakensis unclassified_Oceanospirillales metal polluted marine sediment clone HB2-9-21 DQ334644.1 Impact metals on sediments heavy DQ334644.1 Bacteria; Proteobacteria; Gammaproteobacteria; Oceanospirillales; OCO2 unclassified_Oceanospirillales AY344367.1 Lake Kauhako 30 m clone K2-30- AY344367.1 unclassified_Oceanospirillales 25 positive signals were often found in the epithelia cells of gill from high mortality during winter and spring of every year. An and mantle, and occasionally found in digestive gland cells, but intracellular rickettsia-like prokaryotic parasite was tentatively were not observed in hemocytes, muscles or pericardium. The structural characteristics. The morphology of individual RLOs consistidentified mostly to be of the a roundcausative shape, agent with using occasional histological short andand ultrarod- morphology of RLO inclusions were also identified by HE staining methodDISCUSSION (Figure 5A,5B). shaped morphologies, ranging from approximately 0.58 to 1.20 The Oyster, Crassostrea rivularis Gould (also known as observations reported that RLOs could form basophilic inclusions μm in size and with a smooth trilaminar cell wall [1]. Some Crassostrea ariakensis), mainly distributed in estuary areas, is a major farmed mollusk species in the Hailing Bay area of Guang they could form eosinophilic inclusions [1,24], or even two types Dong province, China. Since 1992, farmed oysters have suffered [17,30] under H&E staining, while other studies reported that

of inclusions can be observed in the same mollusk [23,31,32]. In Ann Clin Cytol Pathol 3(2): 1056 (2017) 6/8 Wu et al. (2017) Email:

Central Bringing Excellence in Open Access this paper, the pathogen was ound only in epithelia cells by in situ hybridization. The RLOs pathogenicity were also different, some studies revealed that RLOs could cause diseases and are responsible for the death of marine mollusks [17,22], whereas other reports

suggested that RLOs only exhibit benign infection [4,30,33,34]. beBy now, used 16S in alignment sequence betweenanalysis has dissimilar been widely microorganism used in bacteria and a variableclassification region as that it has can both allow a highlysequences conserved to be distinguished. region that can In this study, sequence alignment analysis and phylogenetic analysis indicate the RLOs found in oyster is a new kind of bacterium and

most similar to Piscirickettsia salmonis, but the similarity is not can be classified into the gamma subfamily, proteobacteria. It is into the same family. It is representative of a new family in the orderhigh enough of Rickettsiales to support and classification we propose of these the name two kinds Oceanrickettisa of bacteria ariakensis to C. ariakensis RLP. “Oceanrickettisia” is pertaining to the RLO bacterium found in the sea and “ariakensis” is pertaining to this bacterium found in oyster “Crassostrea ariakensis”. CONCLUSION

cells In by conclusion, in situ the final assumed RLOs 16S rDNA sequence Sequencewas reconfirmed alignment and andthe pathogen phylogenetic was found analysis only indicated in epithelia the assumed RLO bacterium hybridization found (ISH) in oyster using C. therivularis specific Gould probes. was most similar to Piscirickettsia salmonis, into a family of gamma proteobacteria. Figure 4 and could be classified in situ hybridization (ISH) staining method. (A) Arrows indicated the ACKNOWLEDGEMENTS Positive signals were detected using specific probes under conditions.positive RLO inclusions found in oyster epithelia. Bar=20um. (B) No signals were detected using none specific probes ISH under the same This work was supported). We by thank research Billy grants Huang from for reviewingScientific Programthis paper of and Zhejiang all the Province members (2004C23041) of the Laboratory and was of Marine supported Life byScience NSFC and (NO. Technology, 31272682 College of Animal Sciences, Zhejiang University for providing the help. REFERENCES 1. Wu XZ, Pan JP. An intracellular procaryotic microorganism associated with the lesion of the oyster Crassostrea ariakensis Gould. J Fish Dis.

2. 2000;Sun JF, 23: Wu 409-414. XZ. Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea

ariakensisIlan P. Epitheliocystis Gould. J Invertebr infection Pathol. in 2004; wild 86: and 77-86. cultured sea bream (Sparus aurata, Sparidae) and grey mullets (Liza ramada, Mugilidae). 3.

4. Aquaculture. 1977; 10: 169-176. organism causing diseases and mortality in Chilean salmonids and its Cvitanich JD, Garate NO, Simth CE. Isolation of a rickettsial-like

Figure 5 confirmationRodger HD, Drinan by Koch’s EM. postulate. Observation J Fish of Dis.a rickettsia-like 1991; 14: 121-145. organism in of RLO parasitizing the epithelial cells of gill of Crassostrea rivularis (A) Numerous intracytoplasmic inclusions (Bold arrows) 5. Atlantic 10 salmon Salmo salar in Ireland. J Fish Dis. 1993; 16: 361- Gould by HE staining method. Note the lytic hollows (small arrows) 369. of the gill epithelial cells by RLO infection (H&E, 500×). (B) Inclusions 6. Fryer JL, Lannan CN. Review: rickettsial and chlamydial infections (arrowheads) in the gill epithelial cells. Note the lytic hollows (arrows) of freshwater and marine fishes, bivalves and crustaceans. Zoo Stud. of the gill epithelial cells by RLO infection (H&E, 1250×). 1994; 33: 95-107. Ann Clin Cytol Pathol 3(2): 1056 (2017) 7/8 Wu et al. (2017) Email:

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7. 21. Harshbarger JC, Chang SC. Chlamydiae (with phages), mycoplasmas,

Fryer JL, Lannan CN. Rickettsila infecions of fish. Annu Rev Fish Dis. 8. 1996;Olsen AB,6: 3-13. Melby HP, Speilberg L, Evensen O, Haastein T. Piscirickettsia and richettsiae in Chesapeake Bay bivalves. Science. 1977; 196: 666- 22. 668.Le Gall G, Chagot D, Miahle E. Branchial rickettsiales-like infection associated with mass mortality of sea scallop, Pecten maximus. Dis salmonis infection in Atlantic salmon Salmo salar in Norway- epidemiological, pathological and microbiological findings. Dis Aquat 9. Org.Mauel 1997; MJ, 31: Miller 35-48. DL. Piscirickettsiosis and piscirickettsiosis-like Aquat Org. 1988; 4: 229-232. York: Adult and juvenile American oysters (Crassostrea virginia) and 23. Meyers TR. Endemic diseases of cultured shellfish of Long Island. New infectionsFederici BA, in Hazardfish: a review. EI, Anthony Vet Microbiol. DW. Rickettsia-like 2002; 87: 279-289.organism causing disease in a crangonid amphipod from Florida. Appl Microbiol. 1974; 24. hardWu XZ, clams Pan (Mercenaria JP. Studies on mercenaria). Rickettsia-like Aquaculture. organism 1981; (RLO) 22: diseases 305-330. of 10. tropical pearl oyster III. Morpholoyg of RLO parasitized in Pinctada

11. Bonami28: 885-886. JR, Pappalardo R. Rickettsial infection in marine crustacea. fucata.Moore Trop JD, Robbins Mar Res. TT, 1997; Friedman 5: 110-117. CS. The role of a rickettsia-like prokaryote in withering syndrome in California red abalone, Haliotis 12. Experientia. 1980; 36: 180-181. 25. Penaeus monodon, associated with Penaeus monodon baculovirus, Anderson IG, Shariff M, Nash G, Nash M. Mortalities of juvenile shrimp, cytoplasmic reo-like virus, and ricketssial and bacterial infections, rufescens. J Shellfish Res. 2000; 19: 525-526. like prokaryote from the scallop Argopecten irradians in China. 26. Li DF, Wu XZ. Purification and biological features of rickettsia- fromLightner Malaysian DV, Redman brackishwater RM, Bonami ponds. JR. Asian Morphological Fish Sci. 1987; evidence 1: 47-64. for a single bacterial etiology in Texas necrotizing hepatopancreatitis in 27. Aquaculture. 2004; 234: 29-40. 13. of a rickettsia-like organism from the oyster Crassostrea ariakensis. Wu X, Sun J, Zhang W, Wen B. Purification and antigenic characteristics Penaeus vannamei (Crustacea: Decapoda). Dis Aquat Org. 1992; 13: 14. 235-239. 28. Dis Aquat Organ. 2005; 67: 149-154.

Kikuchi Y, Sameshima S, Kitade O, Kojima J, Fukatsu T. Novel clade of JacquesKellner-Cousin scallop K,Pecten Le Gall maximus: G, Despres evaluation B, Kahad of M,pro-karyvote Legoux P, Shire diagnosis D, et Rickettsia spp. from leeches. Appl Environ Microbiol. 2002; 68: 999- byal. hybridizatinGenomic DNA with cloning a non-isotopically of rikettsia-like labelled organisms probe and(RLO) polymerase of Saint- 1004.Wang W, Gu Z. Rickettsia-like organism associated with tremor disease and mortality of the Chinese mitten crab Eriocheir sinensis. 15. 29. chain reaction. Dis Aquat Org. 1993; 15: 145-152. Rickettsia-like organism (RLO) in the oyster Crassostrea rivularis Dis Aquat Organ. 2002; 48: 149-153. Wu XZ, Zhang Y. A pair of specific primer for the PCR detection of evidence dune infection rickettsienne chez les Huitres. CR Acad Sc 16. Comps M, Bonami JR, Vago C. Pathology des invertebre’s e Mise en Gould. The People’s Republic of China, CN 100366754C. Feb 6. 17. Paris. 1977; 285: 427-429. fection assocaiated with 30. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular a mass mortality of the sea scallop, Placopecten magellanicus. J Fish Evolutionary Genetics Analysis and sequence alignment. Briefings in Gulka G, Chang PW, Marti KA. Prokaryotic in Bioinformatics.Elston RA. Occurrence 2004; 5: of 150-163. branchial rickettsiales-like infections in two bivalve molluscs, Tapes japonica and Patinopecten yessoensis, with 18. WuDis. 1983; X, Pan 6: 355-364. J. Studies on rickettsia-like organism disease of the 31.

of pinctada maxima pathogen rickettsia-like organism. J Invertebr comments on their significance. J Fish Dis. 1986; 9: 69-71. tropical marine pearl oyster I: the fine structure and morphogenesis in the giant clam Hippopus Hippous associated with rickettsia-like 32. Norton JH, Shepherd MA, Abdon-Nagutt MR, Lindsay S. Mortalities 19. Pathol.Finley CA, 1999; Friedman 73: 162-172. CS. Life history of an exotic sabellid polychaete, organisms. J Invertebr Pathol. 1993; 62: 207-209. the cultured hard clam (Meretrix lusoria Roding) in Taiwan. J Fish Soc Terebrasabella heterouncinata: influence of temperature and 33. Wen CM, Sen T, Kou GH, Chen SN. Rickettsia-like microorganisms in fertilizationWu XZ. Advances strategy. in the J Shellfish research Res. of 2000;marine-cultured 19: 512-513. animal diseases in China. In: Lee C-S, Ventura A, editors. Status of Aquaculture in China. Taiwan. 1994; 20: 347-356. 20. 34. Sparks AK. Synopsis of Invertebrate Pathology Exclusive of Insects. 2003; 29-54. Hawaii, USA in Honolulu: The Oceanic Institute. 1985; Elsevier. Amsterdam.

Cite this article Zhang Y, Fang J, Sun J, Wu X (2017) Identification of Rickettsia-Like Organism (RLO) in the Oyster Crassostrea rivularis Gould. Ann Clin Cytol Pathol 3(2): 1056.

Ann Clin Cytol Pathol 3(2): 1056 (2017) 8/8