Central Annals of Clinical Cytology and Pathology Bringing Excellence in Open Access 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 species 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 proteobacteria. 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 bacteria, 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 the(PBS, 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 bysequences 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 separatelychamber 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