A Novel Screening Method for the Detection of Pseudoalteromonas

Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. b,&Nln,21;Kmih,Mw,Gt,Mtuaa sk,21) h bacterium the 2010); Oseko, adult & of Matsuyama, deaths Goto, as numerous Miwa, such caused Kamaishi, die-offs, which and 2011; , diseases Yang, Nylund, Deng, abalone & Q. several abe, K. causing 2019; pathogens halioticida Hu, the (6 cisella & identified abalone have Li, juvenile Lin, studies of Yang, Previous die-off a Deng, massive 2018) 2020). (K. & the early-June, Li, You, China to & to Luo, led south Lin, (mid-May injury, Liang, Province, heatwave Gu, tissue Fujian 2016; record-breaking obvious in al., a cause 7-month-old) et during not to (Chen However, did world rates which 2015). the survival abalone, epidemic, in al., and Pacific serious country et the performance other You is growth any China 2018; excellent than its in Ke, has to abalone farmed and species abalone more nutrients common this produces of most levels as China the high 2017). its and al., to 2019), due et (Cook, both (Nam worldwide, value species economic aquaculture high important an is abalone The INTRODUCTION 1. wave heat pathogen, tunistic Pseudoalteromonasshioyasakiensis general, in mollusks possibly or KEYWORDS abalone, of is pathogen this opportunistic knowledge, serious our heatwave. a To a is shioyasakiensis. of 2018 shioyasakiensis P. context P. record-breaking in the that the behavior in show with pathogenic to associated opportunistic temperatures report mucosal induced surface first line have sea the first may unprecedented disrupted China The and south mucus, days. in in three to ability heatwave within pathogenic growth for SDCH87 outstanding foot strains strain developed an shioyasakiensis the bacterial had Pseudoalteromonas was in the pathogen to immunity identified opportunistic method exposed This when we novel high bacterial vivo, temperatures. were A Using in high rates at organisms. mortality and marine abalone abalone. vitro of that in immunity healthy verified We innate challenges Bacterial and the abalone. experimental ability. in moribund and mucus attachment no of analysis, of caused of role sequence mucus disease lack important isolation, the this a the on Although and based from pathogens, cells 2018. cultured abalone epithelial in screening and China pedal collected Province, bleached Fujian were exhibited in abalone strains abalone affected Pacific lesions, juvenile external in obvious recorded was disease serious A Abstract 2020 28, August 1 Li Min wave heat discus record-breaking (Haliotis a abalone during Pacific hannai) juvenile of mortality caused mass that the pathogen Pseudoalteromonas opportunistic of emerging detection an the shioyasakiensis, for method screening novel A Luo imnUiest tt e aoaoyo aieEvrnetlScience Environmental Marine of Laboratory Key State University Xiamen 1 n aha Ke Caihuan and , 1 ewiWu Wenwei , hc asdams otlt of mortality mass a caused which , 1 ewiYou Weiwei , 1 , aitsdsu hannai discus Haliotis 1 hxnHuang Shixin , H.tuberculata .gigantea H. 1 nFac n20 Ncls auax aui,& Mazurie, Basuyaux, (Nicolas, 2002 in France in 6 DA uu eitne mrigoppor- emerging resistance, mucus rDNA, 16s , 1 ioi Huang Miaoqin , n20 Bei,Otm aasi Watan- Kamaishi, Ottem, (Brevik, 2005 in 1 igLu Ying , aitsdsu hannai, discus Haliotis iroharveyi Vibrio 1 Xuan , Fran- Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. een edvlpdannivsv,sml,adrpdmto oadesti nweg a.W first We gap. knowledge this address to is method it rapid 2009), and Kushmaro, simple, non-invasive, & a Shnit-Orland developed 2017; especially pathogens, we al., (Carda- of Herein, identification et strains and detection Minniti widespread the for 2010; many approach pathogens. efficient al., between for rapid, opportunistic distinction et more non-obvious a the Choresca develop be As (Cohen to 2017; urgent thus behaviors 2017). al., may pathogenic Egan, et conditional bacteria & Dieguez exhibit Zhong, non-pathogenic to Maturana-Martinez, may and and Bournazos, increases, pathogenic virulent Gardiner, temperature more 2018; especially 2009), al., become conditions, Ke, environmental et to & 2017; in microbes Zhao, Amaro, changes Huang, common & However, Rodriguez-Valera, which Huang, cause 2017). Ghai, by Guo, been (Carda-Dieguez, al., metric 2010; have fish et useful Park, mucus and Minniti a & 2009), and Kushmaro, be Kim, microbes population & might Gomez, and (Shnit-Orland beneficial mucus microbial Choi, corals attachment between host (Choresca, well-balanced relationships in the abalone a Symbiotic bacteria favor in maintain of observed can bacteria. to viability pathogenic animals the host identify Thus, marine the to 2015). of helping Espinosa, secretions microbes, & mucus symbiotic (Allam the specific that of shown growth have studies Previous cuttlefish the inhibited of tissues liquified and uglptoes(nle ta. 06;adthat produced and corals 2016); stressed al., et that Pseudomonasaeruginosa Matz, & (Andleeb shown Genuise, pathogens also Strader, of (Wright, fungal was mucus activity antibacterial Mansouri the It increased that 2016; and 2019); Ghiasi, content 2017). bioactive & Ahmadvand, higher Zorriehzahra, Ding, with & Pourgholam, mucus & experimental Hoseinifar, (Adel, Zhang, in additive pathogens Hajimoradloo, Tang, dietary tested Wang, Taee, against beneficial activity (H. (Sanahuja of with antimicrobial activity higher curves mucus trout showed antiparasitic growth rainbow mucus skin-secreted skin and h the addition, bactericidal In 24 and dose-dependent on 2019). 2019); bass and based sea Mercado, activity) time- and & of lytic robust bream mucus Cortes, (i.e., sea (Valero, skin-secreted growth of growth the bacterial properties mucus microorganismal 2019); skin-secreted limit inhibits the al., or example, also et block immunity, For secreted to 2018). Mucus, innate shown 2014). al., in Buchmann, was et roles 2018; (Bakshani many al., reproduction et plays Bakshani and which 2015; membranes, molecules, Espinosa, antimicro- bioactive mucous & enzymes, release (Allam by proteolytic and lipids synthesize proteins, defense and C-reactive tissues host peptides, immunoglobulins, epithelial the bial mucosal lysozymes, in the lectins, interface lining mucins, important types most including cell the diverse as ( The serve 2007). tissues forces epithelial physical mucosal dynamic the identified against that was known well pathogen is causative It the 2019). which experience al., by previous et process on (Ren the based candidates described pathogen of briefly (Zozaya-Valdes, possible dozens change only among assumed a climate or from authors link and 2018), most to stressors al., However, challenging anthropogenic et as is (Tavares 2015). such behav- individuals it Thomas, pathogenic factors, unhealthy And and & external naturally, from by pathogenic. exist Egan, isolated induced pathogens are opportunistic be be bacteria because can can culturable disease iors strains specific the a bacterial all to numerous not pathogen certain all, methods, to culture known plate is using As deaths. 1998), abalone 2018), maricultural Wakabayashi, al., in & et rates Numata, Hu V.alginolyticus mortality Hasegawa, 2016; high Nishimori, al., caused 2002; et which al., virus, et herpes-like (Nicolas a and 2000); versicolorsupertexta al., et (Friedman bacterium lar of of 2002), Thebault, .dsu hannai discus H. .alginolyticus V. addtseoaitscaliforniensis, CandidatusXenohaliotis C .W,Wn,Ca,&L,21;Y .W,Teg a,21) aeas caused also have 2016), Nan, & Tseng, Wu, S. Y. 2011; Li, & Chan, Wang, Wu, J. (C. n20 Cage l,20) netoso other of Infections 2005). al., et (Chang 2005 in .dsu hannai discus H. n21 a otpsuedss;R .Wn,Yo i,&Wn,21) h intracellu- the 2018); Wang, & Liu, Yao, Wang, X. R. disese; pustule foot (as 2018 in Eiseniafoetida n ekeet on effects weak and tanW1 fe eaaec-nuain(ve l,2019). al., et (Lv co-incubation separate after Wz11 strain V.anguillarum nee sea,21;Bcmn,21;Ln,Hnsn Samuelsson, & Hansson, Lang, 2014; Buchmann, 2012; Esteban, Angeles ´ Sepiapharaonis n20 Fki ath aae 00 aaee l,20) and 2007), al., et Sawabe 2010; Sawabe, & Saitoh, (Fukui, 2007 in xiie togatmcoilatvt gis ua atra and bacterial human against activity antimicrobial strong exhibited Salmosalar MloaoAuy,Tnb alroEcrt,21) and 2014), Gallardo-Escarate, & Teneb, (Maldonado-Aguayo, Staphylococcusaureus ei aspersa Helix togyinhibited strongly hc asdwteigsnrm in syndrome withering caused which a ihlvl fN-yi,adsrn bacteriostatic strong and NK-lysin, of levels high had 2 uu a togatbceileet on effects antibacterial strong had mucus Foh ta. 07.Fnly h mucus the Finally, 2017). al., et (Fuochi Vibrio .harveyi V. pce,including species, V.parahaemolyticus Amphiprionclarkii tanW21adweakly and Wz211 strain .spp H. V.carchariae n2000 in . (Bathige showed .di- H. Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. h oto.TeO au fec itr t60n a eoddatr0 0 0 0 2,ad20min. 240 and 120, these 90, pathogenic; 60, be 30, to 0, likely study after more virulence and recorded resistant for was 28 more candidates nm at were as 200 600 mucus incubated selected of at in and were volume mixture growth strains plate each greater final 96-well with of a a strains value to Bacterial to OD mucus, added The the were control. to the mixtures solutions different bacterial of the washed replicates transferred were by and bacteria determined Collected as USA), 10 0.6–0.8, Spain). Beckman, prepared Tecan, reached We nm spectrophotometer, FSW. 600 Pro with at Pro200 twice value (Infinity (OD) adjusted reader density then microplate optical and a the Kit, when 0.22 Assay phase, Protein a growth BCA through logarithmic Pierce microcentri- a filtered sterile using was was measured a 0.01 process mucus was to to collection Sterile sample transferred mucus The ice. each immediately pedal. on in was concentration kept individual mucus each and surface surface from tube body body mucus fuge abalone collected viscous the The mL from rapidly. mucus 1 Briefly, completed about 2019). al., collected modi- et ultimately some Valero We with 2019; studies, al., et previous Lv following (2.5 2016; collected al., was et mucus (Adel vitro, fications in resistance bacterial investigate To individuals (Xiamen, & healthy (Letunic 2.3Bacterialresistance identifications. and Ltd v5 moribund taxonomic Blastn iTOL between Co., and on bacteria NCBI Bio-Technology 2011) based culturable al., using of determined BORUI et distributions (Tamura were analyzed the by 5.0 in were using MEGA sequencing Differences using purified sequences 2019). constructed being and Bork, DNA was R; tree after System. 1492 phylogenetic A and database Clean-up China). F PCR EzBioCloud (27 and primers the universal Gel against with SV amplified were Wizard products in Promega PCR resuspended were possible. colonies as 200 bacterial soon (SDW), from water DNA deionized 100 genomic sterile the with release twice to washing method and centrifugation cooking the used (Fig. analysis We 6.3 sequence was oxygen gene dissolved rDNA and 16S 35.3, the 2.2 was portable site, salinity a examination. test 7.6, was and using our pathological pH at A.M. for method degC, China) 7:00 fixed plate 1B). 28.3 Instrument, at were was streak 48 AZ measured above temperature the (AZ86031, were for water listed by instrument average farm degC tissues measuring purified each 28 The quality then at water at degC. were parameters multiparameter cultured -80 environmental colonies were aquatic at Individual reduce primary glycerol homogenates to The medium. 15% tissue alcohol culture in and ethyl agar frozen 75% dilutions kept E with mucus gland, washing 2216 hypobranchial The and in gill, collected dissection mantle, contamination. h mucus were pedal, after of diluted the possible homogenized the laboratory, risk (including were as the of the tissues gland) In individuals tops Several digestive healthy analysis. the surface. and further and at body gut, for epithelial moribund the gather refrigeration pedal many from under the to collected As laboratory substrate; tended was 1A). our the abalone to to rates, attachments Affected (Fig. transported mortality pedal and bleached high weak farms. also exhibited extremely affected were and found most morning, cells we March), the the investigation, early in country, of ensuing blocks (since Dongshan some concrete in our rain farms in of During aquaculture lack 100% many China. a reaching in and R. reported P. temperatures were province, high abalones Fujian Pacific continuous juvenile following of 2018, mortalities June mass and May histopathology late and Candidate In isolation, bacterial isolates. outbreak, selected Disease the of 2.1. each METHODS of postulates. AND compared resistance MATERIALS Koch’s as mucus of 2. individuals, tests the diseased for tested on selected enriched we then isolates were Then, the isolates selected individuals. and healthy mucus, to body from bacteria isolated ± . mi egh a bandb ucl isn h nmlwt Lfitrdsaae (FSW). seawater filtered mL 2 with animal the rinsing quickly by obtained was length) in cm 0.2 μ μ / g D n olda 98 at boiled and SDW L μ rti.Ajse ape eesoe t-80 at stored were samples Adjusted protein. L nvitro in × ° o 0mn h uentnswr hnue o C mlfiainas amplification PCR for used then were supernatants The min. 10 for C 10 6 atra(aauae l,21)bsdo o yoer (Cytoflex, cytometry flow on based 2019) al., et (Sanahuja bacteria 3 nvivo in ° .Bceilseiswr olce uigthe during collected were species Bacterial C. atra rwhwsas sesdi 2216 in assessed also was growth Bacterial . μ ffehbceillqi.Bifl,after Briefly, liquid. bacterial fresh of L μ le ormv atra Protein bacteria. remove to filter m ° .FWwr sdas used were FSW C. μ .Three L. Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. rusad1cnrlgop.Ec netdgopwsimre n1o atra ouin:1.0 solutions: bacterial 6 infected of (6 1 groups in 7 immersed was among group divided infected randomly Each were mL group). abalones control 28 140 1 to candidate, and raised each was groups test least To temperature (20 above. at water for the described tanks 0.5 conditions as period, of among laboratory acclimation rate to allocated the acclimatize a After randomly to at experimentation. allowed were before and healthy week abalones FSW a using L challenge, for 10 performed bacterial containing treatment) were to per 2.5 challenges Prior animals length experimental China. shell Province, candidates, average Fujian each old; months of (seven pathogenicity abalone the confirm To (26 isolates temperature survival candidate optimum ofthe the 2.4Thepathogenicity determine to selected also were mucus º to resistance high 28 with at medium liquid E JM181.Rmral,teaudneo strain SDCH97) to of similar and SDCH27, abundance 99.18–99.68% SDCH95, SDCH03, were the data- (SDCH40) Remarkably, SDCH93, SDCH02, abalone EzBioCloud 18891). (SDCH01, SDCH92, healthy the the (JCM abalone SDCH91, from in moribund SDCH90, strain sequences the one in SDCH87, gene and from isolates SDCH31, rDNA isolated two SDCH29, 16S and strains culturable SDCH28, abalone 14 the moribund that with showed the abun- base Comparison in the abalone. abalone. while isolates moribund the (5%), healthy 15 in abalone the included 5%) healthy (to the Pseudoalteromonadaceae drastically to culturable Shewanellaceae, dropped compared Rhodobacteraceae the The as and and (28%) Flavobacteriaceae Vibrionaceae the abalone of the moribund dances of the abundance in exception The greater the individuals. much with moribund was the Pseudoalteromonadaceae, and culturable Flavobacteria- healthy the the Pseudoalteromonadaceae, between of shared Shewanellaceae, Oceanospirillaceae, were (Vibrionaceae, (Alteromonadaceae, Rhodobacteraceae) and only families ceae, family Five healthy the Flammeovirgaceae, Oleiphilaceae). in Ferrimonadaceae, and families Kiloniellaceae, three (Moraxellaceae, and Al- only bacterial Halomonadaceae), NCBI (class five abalone and Proteobacteria identified the moribund phylum We on the the (14%). into in based Bacteroidetes fell families identified by were isolates followed taxonomically 34 most Gammaproteobacteria), and were abalone, and abalone moribund sequences phaproteobacteria moribund and rDNA healthy from 16S isolated both were In The 54 database. abalone. which healthy of strains, from bacterial isolated 93 identified bacteria and isolated ofculturable We distributions the the was in b rate Differences resistance and 3.1 The resistant, 2018). was He, isolate & a/b Weng, RESULTS particular as Dong, 3. the calculated. (Zhu, calculated also exposed which was was was to isolate isolate isolate antibiotics each each the which of of of was to nitrofuran, number (MARI) resistance antibiotics a the index of Drug amphenicol, was number resistance an plates. a antibiotic glycopeptide, agar multiple where a cephalosporins, E The *100, 6 macrolides, 2216 penicillins, 3 polypeptide. 4 on tetracyclines, a China): 3 zones and Ltd., was (BSAC). or aminoglycosides, inhibition Co., 4 Resistance Chemotherapy (I), Reagent the quinolones, susceptibility Microbial Antimicrobial isolate. of 2 (Hangzhou medium each for antibiotics diameters (S), 26 of Society the susceptibility using sensitivity British on assayed antimicrobial antibiotic based the either the (R), by as resistant test recorded of was to guidelines sensitivity method the Antibiotic diffusion to agar according disc assessed the bacteria used postulate, We Koch’s validate was to isolate order candidate testing each In 2.5Antibioticsensitivity of recorded 50). pathogenicity were (LD The group 25 dose abalones. each at moribund lethal in tested the 50% rate also from the mortality identified calculate and and re-isolated symptoms to were Clinical days experiment. 10 the for during daily h 24 every FSW 10 ,o 30 or C, 8 F mL CFU -1 1.0 , ° × ) h Dvlewsrcre vr orfr1 h. 12 for hour every recorded was value OD The C). -1 10 h oto ru a nrae.Tesm oueo ae nec akwsrpae with replaced was tank each in water of volume same The untreated. was group control The . 4 º F mL CFU e a,uigtecntn-eprtr ytm addtswr utrdadwashed and cultured were Candidates system. constant-temperature the using day, per C ° C. ° nodrt xld tan ihhge rwhrtsa hstmeaue Strains temperature. this at rates growth higher with strains exclude to order in C -1 1.0 , × 10 5 F mL CFU -1 1.0 , nvivo in .shioyasakiensis P. ± 4 . m bandfo naaoefr nJinjiang, in farm abalone an from obtained cm) 0.2 × 10 6 F mL CFU -1 E T a 6 ntemoribund the in 26% was (T) SE3 1.0 , × .shioyasakiensis P. 10 7 F mL CFU -1 n 1.0 and , × 10 E (T) SE3 3 º ,28 C, CFU º C, × Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. Asqec iiaiy,w osrce egbrjiigpyoeei re tan DH5adSDCH61 and SDCH25 Strains tree. phylogenetic neighbor-joining a constructed we 15 similarity), sequence the NA of among sequences relationships previously-published the 73–92%. investigate were To strains 16SrDNA isolated on remaining strains based bacterial the the analysis of across 3.5Phylogenetic MARIs common highest from The was the ranging respectively). resistance while abalone. 54%, high, antibiotic cultured and (96%), were multiple (35% from strains that lowest SDCH31 the indicated all strain were results of SDCH27 for These MARIs and recorded 3 The SDCH was 1). of (Table MARI MARIs strains The Only all 96%. tetracycline. to affect and 35% to amikacin, neomycin, able kanamycin, was ceftriaxone, strains chloramphenicol oxacillin, All to 1). (Table resistance nitrofurans complete and showed macrolides, tetracyclines, glycopeptides, polypeptides, 15 aminoglycosides, the of 60% than More temperature. this 15 at ofthe 25 recorded sensitivity at acute were abalone 3.4Antibiotic by deaths any Pacific caused no or towards and were SDCH90 that pathogenicity to challenge, fact show deaths exposure didn’t the the these identical SDCH87 on of Therefore, with strain days eye. associated Importantly, two was naked strain. mortality within the other abalone with occurred was no However, observed mortality deaths infection. abalone were abalone bacterial juvenile injuries all that tissue almost indicated obvious addition, results no In The abalone. SDCH87. dead by 1.0 and obvious caused than moribund as presented (more from well abalones doses samples 5.6 bacterial manner: as mucus was higher dose-dependent mucus SDCH87 to a to exposure of in after resistance value decreased signs outstanding rates clinical survival had same Abalone the SDCH87 abalone. strain on that effects lethal found We results. resistance juvenile bacterial of disease the responses induced The for pathogen, pathogens candidate candidate a as SDCH87, selected optimum3.3 were The SDCH90 3C). and (Fig. increase SDCH87 challenge. Thus, temperature 28 the outbreak. was not disease and SDCH90 mucus-resistance, SDCH90, and the SDCH87, SDCH87 of of both result rates for a growth temperature high were 28 the mucus at that in medium indicating increased liquid SDCH2 SDCH93, 2216E and OD in SDCH92, because SDCH29, rates (p mucus-resistant, first growth including SDCH90 the high considered and had during were SDCH87 strains change SDCH2 for Several not min and did 60 OD SDCH90, within as SDCH87, significantly the unaffected, Strains to considered were min. due SDCH95 mucus-sensitive, 120 (p and considered minute SDCH, of 30 were significantly SDCH92, first resistance SDCH93 differ the SDCH91, not within the and the OD did SDCH40, group, in mucus In mucus SDCH29, decrease the significant min. of SDCH28, the OD 240 Strains In The time. 3A). for mucus-sensitive. over or (Fig. measured unaffected, time mucus-resistant, was over either absorbance were significantly as mixture differ identified not FSW, strains the did and strains mucus group, fresh FSW with co-incubation After 2B). (Fig. abalone, gill healthy the the TheresistanceofPseudoalteromonadaceae from strains In from isolated 3.2 gland. four isolated was digestive was SDCH95 mucus, the SDCH40 strain from from strain isolated foot, (T) was isolated the SE3 SDCH40 from the were strain isolated of and SDCH87) were that, gut, SDCH93) and showed the and analysis SDCH31, SDCH92, Retrospective SDCH91, SDCH29, 2A). (SDCH90, SDCH28, (Fig. abalone SDCH27, healthy SDCH03, the in 2.5% 14 to compared as abalone .shioyasakiensis P. E T tan sltdfo h oiudaaoe ih tan SC0,SDCH02, (SDCH01, strains eight abalone, moribund the from isolated strains (T) SE3 .shioyasakiensis P. .dsu hannai discus H. .shioyasakiensis P. × 10 5 F mL CFU .shioyasakiensis P. .shioyasakiensis P. -1 obceilepsr eeasse ovldt our validate to assessed were exposure bacterial to E T oaaoemcsdffrdntcal Fg B:strains 3B): (Fig. noticeably differed mucus abalone to (T) SE3 tan xiie togrssac opnclis cephalosporins, penicillins, to resistance strong exhibited strains t28 at .shioyasakiensis P. ° Fg ) hsbceilsri a eioae rmthe from re-isolated was strain bacterial This 4). (Fig. C nvitro in ° Fg D,cnitn ihwtrtmeaue during temperatures water with consistent 3D), (Fig. C 5 vial rmteNB slce ae n1SrD- 16S on based (selected NCBI the from available < < .5.SrisSC1 DH,SC2,SDCH31, SDCH27, SDCH3, SDCH1, Strains 0.05). .5,adwti 0mnfrSC2(p SDCH2 for min 90 within and 0.05), strains nvivo in ° u i o xii mucus-resistance, exhibit not did but C tan sltdhri,a ela 60 as well as herein, isolated strains t28 at ° C × 10 4 F mL CFU .shioyasakiensis P. nvitro in -1 .TeLD50 The ). ° bacterial ,based C, < 0.05). Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. 2 i ol o eue sa niao fbceilrssac,bcueatv usacsi h mucus the in substances active because resistance, bacterial of indicator an as used resi- mucus be the not recording Zozaya-Valdes could After ability. 2018; min mucus-resistance al., 120 bacterial et test various (Vezzulli the to of surface in abalone, stance body changes health by and the used accompanied moribund on We usually the mucus environment 2015). are the marine animals al., complex in inter- aquatic the et the microorganisms in in is disease the immersed surface of of directly this Symptoms is as composition invasion, 2018). and pathogen al., conditions, to et external resistant and (Bakshani highly compounded host is was the animals difficulty between aquatic This face most abalone. strain. of juvenile mucus each the body Vibrionaceae of on The 23%, pathogenicity abalone. lesions and the visible moribund (41% of test abalone from lack to moribund identified the the difficult and by 2007), in thus (Ren al., isolated families was contenders et abundant were It possible Sawabe most strains of 1998; respectively). two dozens al., the bacterial among et were 54 from Nishimori Pseudoalteromonadaceae identified Here, 2002; and were al., 2019). pathogens al., et causative et diseases al., Nicolas Fukui the serious abalone 2011; 2010; et how various al., al., Several unclear et of et is 2019). Brevik Kamaishi it outbreaks 2016; (Cook, 2018; but al., to al., et industry led et (Bathige Pacific abalone pathogens Hu has various the Chinese 2010; China, with of the associated in aquaculture been for species previously the high-density consequences have abalone is the fatal Province cultured However, all Fujian with dominant 2019). of advantages, diseases, 90% the (Cook, climatic than is China natural more which its in producing to abalone, area globally, Due abalone producing 2019). of abalone (Cook, producer main worldwide largest the abalone susceptible become farmed only has the were China tested, years, agents recent misuse. chemotherapeutic In antibiotic 15 the indicate all the may of Furthermore, 28 This and, chloramphenicol. behaviors. at antibiotics, to pathogenic multiple exposure to opportunistic of resistant days exhibit 25 were at three to pathogenicity within lost strain SDCH87 mucosa) this Strain (the time. short defense a of within was primary line strain the was that first one suggested foot and the results the gut, that experimental the indicate Our from also damaged. isolated may was was strains of strain from distribution one isolated of This were foot, site gland. strains the digestive eight from the foot. addition, isolated from the In were isolated abalone. to strains bleached moribund four the localized the mucus, and was from the abilities disorder attachment isolated the in species decrease that bacterial abundant the suggested but cells lesions, epithelial external lacked pedal abalone diseased the Here, rtrpr fa ubekascae with associated outbreak an of report first ae nbceilioain euneaayi,adeprmna challenges experimental and analysis, sequence fied isolation, bacterial Therefore, on surface. Based body the on detected by DISCUSSION not invaded 4. were were in virus abalone abnormalities and the no fungi, that identified parasites, observation suspected Histopathological addition, we 6). In (Fig tissues. va- tissue destroying cells analyses other mucosal loose, secretory any Pathological pedal became decreased, tissue the abalone. cells connective of the epithelial integrity moribund and the the increased, of from penetrability secretion cell were tissues epithelial mucus herein occurred, abalone, pedal cuolization moribund isolated the the strains in in that, 17 observed showed of were sequences changes gene Histopathological rDNA MT912618). 16S to MT912612 The (Go numbers cluster. etiology 3.6Histopathology (accession unknown this database of GenBank into mortalities the mass fell strains to of also other investigation submitted the an 2017), of during al., all oysters SDCH29, Pacific et strain from of isolated exception Interestingly, was the which together. With 5). clustered (Fig herein outgroups isolated as used were herein isolated .siyskessstrain shioyasakiensis P. .shioyasakiensis P. Pseudoalteromonas Pseudoalteromonas, neto.Hsooia xmnto hwdta h tutr ftepdlmucosa pedal the of structure the that showed examination Histological infection. DH7a h astv gn fti ubek oorkolde hsi the is this knowledge, our To outbreak. this of agent causative the as SDCH87 tan o 4 i,w otltdta nices nO au after value OD in increase an that postulated we min, 240 for strains .shioyasakiensis P. hc xiie h raetvraini bnac between abundance in variation greatest the exhibited which .shioyasakiensis P. Pseudoalteromonas 6 .shioyasakiensis P. ° ,idctn hthge eprtrsallowed temperatures higher that indicating C, asn ihsme otlt nabalone. in mortality summer high causing DH7truhtepdltissues. pedal the through SDCH87 p tanM40225 (KY229817.1), M14-00202-5E strain sp. tanSC8 togydisrupted strongly SDCH87 strain .shioyasakiensis P. ° nvitro in ,adldt ct mortality acute to led and C, .shioyasakiensis P. and nvivo, in a h most the was eidenti- we strains Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. aymrn aa(auie l,21;Chne l,21;Snesn&Aeadr 00,icuigcorals including 2020), Alexander, & Sanderson in 2018; mortality al., and et disease al., China Cohen of southern et 2016; rates in al., rising Lamb wave et with heat 2019; coincided (Caputi record-breaking al., generally taxa the have et marine to temperatures many due Green ocean mortalities 2020; Changing abalone 2018. Frolicher, organisms in in & marine role impacted Cheung opportunistic significantly an 2016; have played analyses heatwaves al., our Recent conjunction, et 2020). In al., (Caputi 2018). et (K. worldwide Deng climate changing fisheries Q. the K. with and 2019; associated events the al., weather China, destructive et most southern Deng the in of wave one 1D). considered heat (Fig. are Heatwaves the Country April to Dongshan and 28.5 Due Island, to record 2016. Daping March a in from reached late days 25.6 records temperature between 16 to seawater days 2 and increased mean 7 been Meanwhile, 2017 temperatures only has 2018. in air on 2018 maximum days rained June and 17 to It minimum to investigatedaverage years. 2018 compared mortality two March as abalone preceding from 2018, high the period in the during the with (http://www.tianqihoubao.com/lishi/dongshan.html) years, period associated three same country was past this Dongshan 2020) 2018 the in over al., for China that, et data southern show Deng in wave temperature Q. heat K. (Go Air record-breaking 6–13) 2019; a Pacificherein. Similarly, (January al., summertime 2019). 2014 et al., mortality of this Importantly, et Deng summer mass Go, 2019). austral (K. Jenkins, summertime the al., King, during et this 2017; temperatures Go, al., in water Jenkins, et high disease King, with oyster 2017; and coincided al., of die-off established, et et oyster clearly pathogens Go, (Go be causative Jenkins, studies to King, probable previous yet 2017; and the has al., as et M14-00202-5E considered (Go of Pseudoal- stocks pathogenicity were oysters, oyster the a affected However, unaffected the in 2019). in in isolated than al., Australia; was Bay, abundant SDCH87, Cromartys more in with was SDCH87, oysters identity teromonadaceae that Pacific sequence indicate of 100% mortality results showed mass Our which summertime 2010). M14-00202-5E, 2013; al., Strain Imhoff, et abalone. & (Liu bund Wiese, cucumbers Labes, of sea (Goecke, strain and algae Macian, 2003), a 2011), Sitja-Bobadilla, Coyne, identified al., Pujalte, & Verner-Jeffreys, have et 1999; al., Jaffer, (Talpur reports 2016; Ghiorse, et Schroeder, crabs & few Riquelme, Nagel al., 2007), (Nelson a Garay, 2016; fish & et Only & including Bauer, Leyton, Alvarez-Pellitero, (Chi 2018). organisms, & marine Pespeni, Sayes, cucumbers Kirby, to & sea 2016; Naidoo, pathogenic al., (Lloyd (Albakosh, 2013), as al., stars et algae Hoj, et sea (Amoah marine & and shrimp (Mladineo 2004), 2012), Baillie, 2016) Birkbeck, fish Pereg, al., & 2012), et Hall, Bricknell, al., Sun Shields, (Goulden, et et 2015; lobster Rosado Bazire, Zheng 2014), bivalves & 2018; marine 2014; al., 2019), Dufour, Permata, 2012), et al., Paillard, Kushmaro, & et Pham Rodrigues, Rochard, & 2019; Sabdono, Offret, 2014; Sivan, 2019; Muchlissin, al., Shnit-Orland, Chevalier, et 2014; Le 2015; & (Desriac Radjasa, al., Fleury, & Mounier, et Jegou, Amelia, (Offret, (Moree Kartika, abalone Sawonua, coral Sabdono, in 2019; probiotics al., as act nerally bacteria. pathogenic of new identified of Species effectively identification method the for our framework that a non-invasive provide demonstrated for rapid, also we results a results Here, our is the organisms; pathogens. mucus pathogenic in with epidemic-causing composition identify Consistent microbial to 2019). in way changes of al., monitoring liquids et Critically, Postulates. both (Lv Koch’s with mortality co-incubation higher h caused cus et 2 and Jung-Schroers 2017; a Wz21 al., after strain et (Fuochi rates mucus 2019). survival in ability al., high growth collagenase well et as Wright and is manifest mucus 2019; 2008), can It to al., it resistance al., data. and bacterial et including important, growth Therefore, immunity, (Ridgway extremely 2019). bacterial host Mukherjee, is proteases with on & extracellular Hazra, associated based Gachhui, 2015), molecules Bhattacharya, pathogenic (Bhattacharya, al., effector (Guo as resist et previously SDCH87 can (Choudhury shown factors strain metalloproteases was virulence identified as bacterial we time, the over Here, known nutrients 2009). bacterially-available al., into et down break eventually might tanW1,tehg ahgnct fsri DH7wssbeunl eosrtdacrigto according demonstrated subsequently was SDCH87 strain of pathogenicity high the Wz11, strain Pseudoalteromonas P.shioyasakiensis ssrosptoe.W sltdti tanfo h ea uu fmori- of mucus pedal the from strain this isolated We pathogen. serious is , .alginolyticus V. n ftems omnbceilgnr ntemrn niomn,ge- environment, marine the in genera bacterial common most the of one , nvivo in xeiet t25 at experiments tanW1,wihhdsrn eoyi ciiy exhibited activity, hemolytic strong had which Wz11, strain 7 ° n 28 and C º º .pharaonis S. ,acrigt nulsatemperature sea annual to according C, n 32.3 and C ° ugse that suggested C º ,rsetvl,i aeMay late in respectively, C, Pseudoalteromonas oprdwith compared .shioyasakiensis P. º amrthan warmer C Vibrio .alginolyti- V. .harveyi V. species species Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. nle,S,Ea,M,Aa,U . l,S,Kyn,A,Saqe . aa,A 21) nvtosreigof screening vitro In (2016). A. Zafar, & I., 29 Shafique, Sciences, A., Pharmaceutical Kiyani, of S., Dietary of Ali, extracts (2019). A., solvent H. U. and mucus X. Awan, shrimp, M., Dong, Ejaz, white . S., Pacific . Andleeb, . of H., confrontation Q. disease Yang, and Y., vannamei S. response, Chi, immune S., microflora, Zhang, morphology, P., probiotic B. Tan, of C., supplementation Q. Huang, In K., mollusks. Amoah, in immunity Mucosal (2015). com- P. bacterial E. 325-370). epiphytic Espinosa, of & Identification B., Allam, (2016). R. alga Bauer, brown & doi:10.1007/s10811-015-0725-z B., the 1891-1901. Kirby, with K., associated R. munities Naidoo, A., M. Albakosh, biochemical and ( Immunological trout doi:10.1016/j.fsi.2016.05.040 against - rainbow piperita Hemato Mentha in (2016). with survival supplemented M. and diet Ghiasi, activity, & antibacterial J., skin Zorriehzahra, parameters, R., Pourgholam, M., declare. Adel, to interest of conflict no have REFERENCE request. manuscript upon the author in corresponding authors the the from All available are study INTEREST this OF of CONFLICT findings the support that from data pathogenic The officers not suffering. quarantine were animal by here avoid collected to analyzed STATEMENT professionally, treatment AVAILABILITY isolates DATA or were, page, all monitoring abalone guidelines as their China author from during required journal’s samples abalones was the of biological restrained in approval noted and ethical as Program humans No journal, the to to. of Development adhered policies been ethical and have the that confirm Research authors Fuji- The and Key 31872564), National and STATEMENT (2019N0001). (U1605213 ETHICS Project the China T of & by S Foundation Provincial Science an supported Natural National was (2018YFD0901401), work in increases This and pathogens opportunistic of emergence ACKNOWLEDGEMENTS global pathogen, recent host, the among events. interactions responsible to disease complex the led mass promote the have to consider Importantly, that must general. environment we in industry, and mollusks even aquaculture or the abalone, al., of pacific et development cultured (Larsen of climate organisms health of the continuing aquatic the abundance with of enhance of that, high health decreasing imply may and the may prevalence and challenge temperatures This The will metabolism 2018). 2018). Elevated pathogens Baden, increasing opportunistic 2019). & emerging by Hernroth al., more 2018; microorganisms change, al., et marine et (Zhang (Cohen many time disease of generation ulcer adaptability 2017 rainbow skin summer and during in caused pathogenicity mussels mortality rockfish fan of Acute black mortality emerging 2010), 2019). of unexpected al., an the Labbate, with while et with & associated 2018), (Fukui associated was Singh, Seymour, gastropods & was marine Jenkins, Chandra, months 2001), Sahoo, King, summer Mallik, al., 2019; the et al., Porter during 2002; et trout al., (Green et oysters Harvell and 2007; Garrabou, & (Bally . ih&Sels muooy 87 Immunology, Shellfish & Fish alsoiimpinnae Haplosporidium iei foetida Eisenia ailscoagulans Bacillus 3,969-977. (3), pahiimrugosum Splachnidium Pnrs ta. 09.Fnly neegn umrpathogen summer emerging an Finally, 2019). al., et (Panarese gis ua atra n uglpathogens. fungal and bacterial human against .shioyasakiensis P. 9-0.doi:10.1016/j.fsi.2019.02.029 796-808. , esnaruckeri Yersinia TC75,ipoe h rwhpromne intestinal performance, growth the improves 7050, ATCC 8 a ersn oe,ipratbiomarker important novel, a represent may . ih&Sels muooy 55 Immunology, Shellfish & Fish . ora fApidPyooy 28 Phycology, Applied of Journal atccu garvieae Lactococcus nohnhsmykiss Oncorhynchus uoa elhi Aquaculture in Health Mucosal aitnJournal Pakistan neto (Shahi, infection olwn the following ) Litopenaeus 267-273. , (pp. (3), Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863400.08792481 — This a preprint and has not been peer reviewed. Data may be preliminary. huhr,J . rmnk . ese,N . lwly,L . ahu,R,&Mkeje .(2015). 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