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RESEARCHOpenAccessAhereditarydispositionforbovineperipheralnervesheathtumorsinDanishHolsteincattleAnetteBGrossi1,5,JrgenSAgerholm2,KnudChristensen3,HenrikEJensen1,PállSLeifsson1*,ChristianBendixen4,PeterKarlskov-Mortensen3andMereteFredholm3AbstractBackground:Peripheralnervesheathtumors(PNSTs)arefrequentlyfoundinDanishcattleatslaughter.
BovinePNSTsshareseveralgrossandhistopathologicalcharacteristicswiththePNSTsinhumanswithheritableneurofibromatosissyndromes.
TheaimofthepresentstudywastoinvestigateapossiblehereditarydispositiontoPNSTsindairycattlebystatisticalanalysisperformedondatafrom567cattlewithPNSTs.
Furthermore,apreliminarygenome-wideassociationstudy(GWAS)wasperformedonDNAisolatedfrom28affectedand28non-affectedHolsteincowstoidentifylociinthebovinegenomeinvolvedinthedevelopmentofPNSTs.
Results:PNSTsweresignificantlymorecommonintheDanishHolsteinbreedthaninotherbreedswith0.
49%ofDanishHolsteinsslaughteredduringaneight-year-periodhavingPNSTs.
PNSTsalsooccurredsignificantlymorefrequentlyintheoffspringofsomespecificHolsteinsires.
ExaminationofthreegenerationpedigreesshowedthatthesesiresweregeneticallyrelatedthroughawidelyusedUSHolsteinsire.
ThePNSTsincludedinGWASwerehistologicallyclassifiedasneurofibroma-schwannoma(43%),schwannoma(36%)andneurofibroma(21%)andderivedfromHolsteincowswithmultiplePNSTs.
AsingleSNPonchromosome27reachedgenome-widesignificance.
Conclusions:GrossandhistologicalcharacteristicsofbovinePNSTsarecomparabletoPNSTsinhumans(schwannomatosis).
DanishHolsteinsaregeneticallydisposedtodevelopPNSTsbuttheexaminedmaterialsareinsufficienttoallowdeterminationofthemodeofinheritance.
Keywords:Cattle,Genetics,Genomewideassociationstudy,Neoplasms,Neurofibroma,Neurofibromatosis,SchwannomaBackgroundSeveralabattoirsurveysofneoplasmsincattlehaveshownthatbovineperipheralnervesheathtumors(PNSTs)areamongthethreemostcommonneoplasmsincattle[1-5].
BovinePNSTsrarelygiverisetoclinicalsignsandaremostcommonlyfoundinthebrachialnerveplexus,heart,andintercostalandmediastinalnervesofoldcowsatthetimeofslaughter[3,4,6,7].
Dependingontheirhistologicalandimmunohistochemicalcharacteristics,benignbovinePNSTscanbeclassifiedintoschwannoma,neurofibroma,andhybridneurofibroma-schwannoma[7].
Thehistomor-phologicalandimmunohistochemicalcharacteristicsofthesubtypesofbovinePNSTsarecomparabletohumanPNSTswithneurofibromatosissyndromes1and2,andschwannomatosis[7].
Inhumansneurofibromatosis1and2(NF1andNF2)areautosomaldominantgeneticdisor-ders,andhalfofallcasesareinheritedfromaparentwithNF1orNF2[8,9].
Incontrast,morethan75%oftheschwannomatosiscasesoccursporadically[10].
NF1ischaracterizedbytheonsetofmultipleclinicalsignsincludingthedevelopmentofmultiplecutaneousneuro-fibromas[11].
OneofthekeystepsintheformationofneurofibromasislossofNF1tumorsuppressorgenefunc-tioninSchwanncells.
TheNF1geneproductneurofibro-minfacilitatestheinactivationofRasproteinsthatregulatecellularresponsessuchasmitogenesisandmigration[11].
NF2islesscommonthanNF1andischaracterizedbyschwannomasofthecranialandspinalnerveroots.
PatientswithNF2typicallypresentwithuni-orbilateralvestibularschwannomainadditiontoothertumortypeslikemeningiomaandglioma[8].
TheNF2gene,whichisinactivatedinpatientswithNF2,encodesaproteincalledMerlinofunknownfunction[8].
Finally,multiple*Correspondence:ple@sund.
ku.
dk1DepartmentofVeterinaryDiseaseBiology,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Ridebanevej3,1870FrederiksbergC,DenmarkFulllistofauthorinformationisavailableattheendofthearticle2014Grossietal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Grossietal.
ActaVeterinariaScandinavica2014,56:85http://www.
actavetscand.
com/content/56/1/85schwannomas,hybridneurofibroma-schwannomasandsometimesneurofibromasinperipheralandcranialnervesarecharacteristicofschwannomatosis[12,13].
In40-50%ofthefamilialandin10%ofthesporadiccasesofschwan-nomatosisthetumorsuppressorgeneSMARCB1isinvolvedinthepathogenesis[10].
AdisorderresemblingNF1hasbeendescribedinfourHolsteincowsfromthesameherd[14].
Thecowshadmultiplecutaneousneurofibromasandthreeofthemwerefromthesamesirelineage.
Thesamealleleofaninform-ativepolymorphismattheNF1locuswasdetectedintwoofthecowsandtheirsire[14].
TheobservationthatseveralanimalsfromthesameherddevelopPNSTshasledtothespeculationthatbovinePNSTsmightbeinducedbyavirus[15,16].
Inaddition,someauthorshavefoundvirus-likeparticlesinSchwanncellsandfibroblastsbyultrastructuralexaminationofbovinePNSTs[16-18].
However,attemptstoconfirmtheviraloriginoftheparti-clesbyvirusisolation,immunohistochemistryoranimalinoculationshavebeenunsuccessful[16-18].
Theaimofthepresentstudywastoinvestigateapos-siblehereditarydispositiontoPNSTsincattle.
Thereforeweperformedstatisticalanalysesondatafrom567slaughtereddairycattlediagnosedwithPNSTsatpostmorteminspection.
Moreover,apreliminarygenomewideassociationstudy(GWAS)on28cowswithconfirmedPNSTsand28cowswithnopathologicalevidenceofPNSTswasperformedtoidentifylociinthebovinegenomeinvolvedinthepathogenesisofPNSTs.
MethodsThetissuespecimensformicroscopicalexaminationandgeneticanalysisderivedfrom28slaughteredHolsteincowsdiagnosedwithPNSTsatthepostmorteminspection.
Attheabattoir,specimensofneoplastictissuefromeachanimalwerefixedin10%neutralbufferedformalin.
Inaddition,samplesfromthespleenandPNSTswerestoredat–20°C.
Spleensamplesof28unaffectedHolsteincowsaged≥9yearswerealsofrozenandusedascontrolsinthegeneticanalyses.
CarcasseswithPNSTsunderwentdeboningtodiscloseallneoplasms,andtheeartagnumberoftheanimalandthelocationofthePNSTswererecorded.
HistologicalandimmunohistochemicalexaminationsTheformalinfixedspecimenswereprocessedconvention-allyandembeddedinparaffin.
Fromeachsample,2–3μmsectionswerecutandstainedwithhematoxylinandeosin(HE)forhistologicalassessment.
Forimmunohistochemis-try,sectionsweremountedonadhesive-coatedslides(SuperfrostPlus;Menzel-Glazer,Braunschweig,Germany),processedthroughxylene,andrehydratedinethanol.
Anti-genretrievalwasdonebyboilinginamicrowaveoven(700W)twicefor5mininTris-EDTAbuffer(1.
21gTRISbase[A1379;Applichem,Darmstadt,Germany]and0.
372gEDTAbuffer[8418;Merck,Darmstadt,Germany]to1literofdistilledwater),pH9(S100andNF);or0.
01Mcitratebuffer,pH6(CNPase).
Endogenousperoxidaseandunspecificproteinbindingwereblockedwith0.
6%(v/v)H2O2inTBS(pH7.
6)for15minatroomtemperatureandwithUltraVblock(LabVision,ThermoFisherSci-ence,Fremont,CA,USA)for5minatroomtemperature,respectively.
Theslideswereincubatedfor24hat4°CwithprimaryantibodiestoCNPase(clone11-5B,Sigma-Aldrich)diluted1:800;S100(polyclonal,DakoCytomation)diluted1:5000;andNF(polyclonal,AbDSerotec)diluted1:6000.
ThedetectionsystemUltraVisionONE,HRPpoly-merwasappliedinaccordancewiththemanufacturer'sinstructions(LabVisionThermoFisherScientific).
Thechromogenwas3-amino-9-ethylcarbazoloe(AEC-red)(LabVisionThermoFisherScientific),andthesectionswerecounterstainedwithMayer'shematoxylin.
Slidesweregiventwo5minwashesinTBSatpH7.
6beforeadditionofeachreagent.
Normalbovineperipheralnervetissuewasusedaspositivecontrols.
Negativecontrolswiththeprimaryantibodyreplacedby1%bovineserumalbumininTBS,5%normalswineseruminTBS,orwithanonsensemonoclonal(matchingisotype)orpolyclonalantibodyofthesameconcentrationastheprimaryantibodywereruninparallel.
StatisticalanalysisIntheperiod2003–2010,theuniqueeartagnumberofallcattlediagnosedwithPNSTsattheDanishCrownslaugh-terhouseinTnder,Denmarkwasregistered.
Atotalof669animalswereregisteredashavingPNSTs,andthefollowingdatawereobtainedfromtheDanishCattleDatabaseonallanimals:age,sex,breed,herdlocationatbirth,herdlocationimmediatelybeforeslaughter,sire,paternalandmaternalgrandsires,dateoflatestcalving,numberoflactations,detailsofmilkproduction(volume,fatandprotein)duringthepreceding305days,andweightofcarcass.
DataoncattlewithPNSTswerecomparedtodataonallcattleslaughteredattheabattoirin2003–2010(n=699,116).
Thestatisticalanalyseswererestrictedtoanimalsaged5–12yearsbeingoftheHolstein,RedDanishDairyandJerseybreeds(n=144,432).
However,allbreedswereincludedinthestatisticalanalysisoftheeffectofsex.
Whenanalyzingtheeffectofsire,theanalyseswerelimitedtoHolsteins,duetothelowoccurrenceofPNSTsintheotherbreeds,andthedatasetwasreducedtosireswithmorethanfiveaffectedoffspringaged5–12years.
Furthermore,theageoftheoffspringwasincludedinthemodelasasignificantdifferencecouldbeduetothelongerlifeexpectanceoftheoffspringofsomebullsincomparisonwithotherbulls.
DatawereanalyzedbytheChi-squaretest,Fisher'sexacttest,ortheGeneralLinearModelprocedure(SASInstitute,Cary,NC,USA).
TheGrossietal.
ActaVeterinariaScandinavica2014,56:85Page2of6http://www.
actavetscand.
com/content/56/1/85GLMprocedurewasusedapplyingtheSASmodelstate-mentsweremodelPNST=agesire,andmodelPNST=agegrand-sire,where'age','sire'and'grand-sire'weredefinedasclassvariables.
Additionally,amodelincludingthepro-ductiontraitsmilkyieldandweightatslaughterwasapplied.
Genome-wideassociationstudyGenomicDNAfrom28casesand28controlswasisolatedfromthespleenusingthesaltingoutprocedure[19]withmodifications.
Briefly,cellsfrom0.
1-0.
5gtissuewerelysedin500μLlysisbuffer(100mMTris,5nMEDTA,200mMNaCland0.
2%SDS)and10μLproteinaseKfor24hat55°C,DNAwasprecipitatedwith500μLisopropa-nolandthepelletwaswashedwith70%ethanolanddis-solvedin200μLTEbuffer(10mMTris,1mMEDTA),DNAwasprecipitatedwith20μL5MNaCLand600μL100%ethanolanddissolvedin100μL.
GenotypingwasperformedonDNAsamplesdilutedto50μg/μLatGenoScanA/S,Tjele,DenmarkusingtheBovineSNP50BeadChip.
GWAwasperformedusingPLINK[20]andallmarkersweresubjecttostrictqualitycontrol;onlySNPswithaminorallelefrequency>5%,acallrateof>90%andinHardy-Weinbergequilibriumincontrols(P=0.
05)wereincludedinsubsequentanalysis.
Allsampleshadlessthan10%missinggenotypecalls.
Thethresholdforgenome-widesignificancewassetbyapermutationtestusing100,000permutations.
Multidimensionalscaling(MDS)analysiswascarriedoutusingPLINK[20]andusedtoassesspopulationstratification.
ResultsGrossandhistologicalexaminationsThe28HolsteincowsincludedinthegeneticanalysisallpresentedwithmultiplePNSTsthatwereconfinedtothebrachialnerveplexus(21),intercostalnerves(21)(Figure1),mediastinalnerves(16),theheart(9),spinalnerveroots(2),andinlymphnodes(1).
ThePNSTswereclassifiedinaccordancewiththeclassi-ficationcriteriaproposedbyNielsenetal.
[7]ashybridneurofibroma-schwannoma(12/28),schwannoma(10/28)andneurofibroma(6/28).
Inschwannomas,theAntoniApatternwaspredominantwithcloselypackedspindle-shapedcellsarrangedinwhorls,bundlesorpalisadeswithVerocaybodies.
SmallerareaswithAntoniBpatternchar-acterizedbyroundedandlooselyarrangedcellswerealsoseen.
Inschwannomas,strong,diffuseimmunoreactivityforCNPaseandS100wascharacteristic.
Theneurofibromasweremoreheterogeneous,lesscellular,andwithcellsorga-nizedmorelooselyintobundlesorshortinterwovenfasci-cles.
TheimmunolabelingforCNPaseandS100wasfocalandonlyseeninsomeoftheneoplasticcells.
NFpositiveaxonsweremainlyseeninneurofibromasandareasofneurofibroma.
Hybridneurofibroma-schwannomaswerecomposedofbothschwannomaandneurofibroma.
Inthemajorityofthehybridtumorstheschwannomacomponentwaspredominating.
StatisticalanalysesPNSTswerediagnosedin669slaughtercattle(0.
09%)during2003–2010.
Ofthese,562wereDanishHolsteinsandfivewereRedDanishDairycattle.
Theremainingcaseswereeitherbeefcattleorcrossbreds,whilenocaseswererecordedinJerseycattle.
ThestatisticalanalyseswererestrictedtotheHolsteinandRedDanishDairybreeds(n=567)whenrelevant.
BreedTheanimalssurveyedincludedthethreemostcommonDanishdairybreeds:115,248Holsteins,ofwhich0.
49%hadPNSTs;10,045RedDanishDairy,ofwhich0.
05%hadPNSTs;and19,139Jersey,ofwhichnonewereaf-fected.
Thus,PNSTsweresignificantlymoreprevalentintheHolsteinbreedthanintheotherbreeds(x2=223.
62,df=2,P98%and40,962SNPspassedqualitycheck.
All56individualspassedqualitycontrol.
AfullmodelassociationtestwithcalculationofexactP-valueswasperformedinPLINKusingtheop-tions'–model'and'–fisher'.
FourSNPswereidentifiedwithpointwiseP-values(EMP1)of0.
000999(Table1).
OnlyoneSNP(HAPmap49086-BTA-22323)reachedgenome-widesignificanceaftergenomiccorrection(EMP1GC)andaftercorrectinginitialrawP-valuesformultiplehypothesestestingbypermutation(EMP2).
EMP1GC=7.
88*106;EMPBonferroni=0.
049;EMP2=0.
02298;BenjaminiHochbergFalseDiscoveryRate(FDRBH)=0.
049.
TheneighboringSNP(ARS-BFGL-NGS-65900)alsostandsoutcomparedtootherSNPsonchromosome27butdidnotreachgenomewidesignifi-cancelevel(Figure3).
HAPmap49086-BTA-22323islocatedat20.
9Mbonchromosome27(UMD3).
ThisSNPwasidentifiedundertheallelicmodelandnoothermodelresultedinsignificantassociations.
Thegenotypedistributionofthechromosome27SNPreachinggenome-widesignificancewassixAGand22GGanimalsamongthecontrolsand10AA,10AG,8GGamongthePNSTcases.
DiscussionSeveralpointsofresemblancetopatientswithschwan-nomatosiswereobserved.
Asinhumans[12],theanimalspresentedwithmultipleschwannomas,hybridFigure2AssociationbetweenageandprevalenceofperipheralnervesheathtumorsinDanishdairycattle(n=567)(Holsteins:n=562;DanishRed:n=5).
Table1P-valuesofSNPsreachinggenome-widesignificanceCHRSNPEMP1EMP227Hapmap49086-BTA-223230.
0009990.
0229827ARS-BFGL-NGS-659000.
0009990.
0999CHR:chromosomeno;SNP:Singlenucleotidepolymorphism;EMP1:InitialempiricalP-values;EMP2:EmpiricalP-valuesaftercorrectionformultiplehypothesistestingbypermutation.
Grossietal.
ActaVeterinariaScandinavica2014,56:85Page4of6http://www.
actavetscand.
com/content/56/1/85neurofibroma-schwannomasandsometimesneurofibro-masconfinedtospinalnerverootsandperipheralnerves.
Themajorityofthehumancasesofschwanno-matosisaresporadicandonlyinabout15-25%ofthepatients,thediseaseisinheritedasanautosomaldomin-anttrait[10].
InDanishHolsteincattle,theincidenceofPNSTswassignificantlyhigherthaninthebreedsRedDanishDairyandJersey,whicharecomparableregard-ingmanagementpracticeslikeforexamplecullingage.
ThisindicatesahereditarydispositiontoPNSTsinHolsteins.
AllcattleincludedinthestudywereslaughteredatoneabattoirinthesouthernpartofDenmark.
WecanthereforenotextrapolatetheprevalencetotheentireDanishcattlepopulation.
AviraletiologyofPNSTsincattlehasbeenproposed[17,18]buttherearenodatainourstudythatsupportsthathypothesis.
Significantbreeddifferenceswereobserved,whichpointsagainstaninfectiousetiologytakingintoconsiderationthatthethreemajordairybreedsareheldundercomparableconditionsandthataffectedcattlederivedfrom248dif-ferentfarmsandwithoutvariationsovertime.
Inhumans,sporadicschwannomasoccurinindividualsofallageswithapeakincidencebetweenthethirdandsixthdecades;whereas,themajorityofindividualswithfamilialschwannomatosispresentswithclinicalsigns,usuallychronicpain,atayoungerage(secondandthirddecade)[21].
Incomparison,theincidenceofPNSTsincattleincreasedwithage.
Furthermore,therewasnoeffectofPNSTsonweightatslaughterandmilkyield,whichwouldbeexpectedinanimalswithchronicpain.
AnalysisofpedigreedatainthepresentstudyfailedtoshowaclearMendelianinheritanceofPNSTs.
However,astatisticalsignificanteffectofsiresandmaternalgrand-sireswasfound.
PhenotypicinformationonPNSTinthegrandsireswasnotavailable.
Someofthesireswerera-thercloselyrelatedthroughacommonancestor,butthismaysimplybebychance,assomebreedinglinesareveryfrequentlyrepresentedintheHolsteinbreedworld-wide.
AstheincidenceofPNSTsincreaseswithage,itispossiblethatsomeoftheanimalsincludedinthestatis-ticalanalyseswouldhavedevelopedPNSTsifallowedtolivelonger.
Thus,aMendelianinheritancemighthavebeenidentifiedifalltheanimalshadbeenslaughteredattheageof12years.
TheGWASidentifiedasingleSNPatchromosome27associatedwithPNST.
Additionally,thisSNPandtheneighboringSNPsweretheonlytwoSNPsinthestudythatstoodoutfromtherest.
ThetestforassociationtoeveryotherSNPshadap-valuecloseto1.
Theresult,however,stillhastobeinterpretedwithcautionasaGWASwithsofewanimalshasverylowpower.
Hence,theassociationreportedherecanonlybetakenasanin-dicationonthepresenceofageneticlocusonchromo-some27affectingPNSTinHolsteindairycattleandmustbefollowedupbymorepowerfulstudies.
ConclusionsBovinePNSTsmorphologicallyresembleschwannomato-sisinhumans.
DanishHolsteincattlehaveasignificantlyhigherprevalenceofPNSTsthantheotherDanishdairybreedsaswellasthereweresignificanteffectofsireandgrandsireontheprevalenceintheHolsteinbreedthusin-dicatingahereditarydisposition.
However,moreanimalsneedtobeexaminedinordertodeterminethemodeofinheritanceandtheinvolvementofspecificlociorgenesinthepathogenesisofbovinePNSTs.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Figure3Manhattanplotofgenome-wideassociationstudyresults.
X-axis:Positiononbovinechromosome27inmegabasepairs(Mbp)accordingtotheUMD3assembly.
Y-axis:log(EMP2),whereEMP2isthegenomewidecorrectedempiricalP-valuedeterminedbasedon100,000permutations.
Thehorizontallineindicatesthegenomewidesignificancethreshold.
OneSNP,HAPmap49086-BTA-22323at20.
9Mb,exceedsthegenomewidesignificancelevelandtheneighboringSNPapproachesthegenomewidesignificancelevel.
AllotherSNPsonchromosome27andintherestofthegenome(datanotshown)hasa-log(EMP2)closeto0.
Grossietal.
ActaVeterinariaScandinavica2014,56:85Page5of6http://www.
actavetscand.
com/content/56/1/85Authors'contributionsABGparticipatedinthestudydesignandcoordinationcarriedoutthehistologicalandimmunohistochemicalexamination,performedtheDNAextraction,participatedinthestatisticalanalysesofpedigreeandproductiondata,anddraftedthemanuscript.
JSAparticipatedinthestudydesign,acquiredthedataforstatisticalanalysis,andhelpedininvestigatingthepedigrees.
HEJandPSLconceivedofthestudyandhelpedinestablishingcontacttoexternalcoworkersandabattoirs.
KCparticipatedthestatisticalanalysesofpedigreeandproductiondata.
PKMandCBcarriedouttheGWAS.
MFcontributedtothestudydesignandparticipatedintheDNAextractionandGWAS.
Allauthorsparticipatedinwritingofthemanuscriptandapprovedthefinalmanuscript.
AcknowledgementWethankForsgslederR.
NrtoftThomsensFondenforfunding,whichmadeitpossibletoacquirethetissuesamplesusedintheGWAS.
WearegratefultoMinnaJakobsenandAnneStrandsby,DepartmentofVeterinaryClinicalandAnimalSciencesfortheirvaluablehelpinDNAextraction.
WethankBetinaGAndersen,LisbetKirboeandHanneHMller,DepartmentofVeterinaryDiseaseBiologyfortheirhelpintissueandslidepreparation.
AspecialthankstoRenateJütting,DCTnderforheroutstandingregistrationsofneoplasiaincattle,whichprovidedvaluabledataandgavetheinspirationforthestudy.
Authordetails1DepartmentofVeterinaryDiseaseBiology,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Ridebanevej3,1870FrederiksbergC,Denmark.
2DepartmentofLargeAnimalSciences,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Dyrlgevej68,1870FrederiksbergC,Denmark.
3DepartmentofVeterinaryClinicalandAnimalSciences,FacultyofHealthandMedicalSciences,UniversityofCopenhagen,Grnnegrdsvej3,1870FrederiksbergC,Denmark.
4DepartmentofMolecularBiologyandGenetics,FacultyofScienceandTechnology,UniversityofAarhus,BlichersAllé20,8830Tjele,Denmark.
5Presentaddress:EllegaardGttingenMinipigsA/S,SorLandevej302,4261Dalmose,Denmark.
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doi:10.
1186/s13028-014-0085-8Citethisarticleas:Grossietal.
:AhereditarydispositionforbovineperipheralnervesheathtumorsinDanishHolsteincattle.
ActaVeterinariaScandinavica201456:85.
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pacificrack:VPS降价,SSD价格下降

之前几个月由于CHIA挖矿导致全球固态硬盘的价格疯涨,如今硬盘挖矿基本上已死,硬盘的价格基本上恢复到常规价位,所以,pacificrack决定对全系Cloud server进行价格调整,降幅较大,“如果您是老用户,请通过续费管理或升级套餐,获取同步到最新的定价”。官方网站:https://pacificrack.com支持PayPal、支付宝等方式付款VPS特征:基于KVM虚拟,纯SSD raid...

Vultr再次发布充值多少送多少活动

昨天我们很多小伙伴们应该都有看到,包括有隔壁的一些博主们都有发布Vultr商家新的新用户注册福利活动。以前是有赠送100美元有效期30天的,这次改成有效期14天。早年才开始的时候有效期是60天的,这个是商家行为,主要还是吸引到我们后续的充值使用,毕竟他们的体验金赠送,在同类商家中算是比较大方的。昨天活动内容:重新调整Vultr新注册用户赠送100美元奖励金有效期14天今天早上群里的朋友告诉我,两年...

Sharktech($49/月),10G端口 32GB内存,鲨鱼机房新用户赠送$50

Sharktech 鲨鱼机房商家我们是不是算比较熟悉的,因为有很多的服务商渠道的高防服务器都是拿他们家的机器然后部署高防VPS主机的,不过这几年Sharktech商家有自己直接销售云服务器产品,比如看到有新增公有云主机有促销活动,一般有人可能买回去自己搭建虚拟主机拆分销售的,有的也是自用的。有看到不少网友在分享到鲨鱼机房商家促销活动期间,有赠送开通公有云主机$50,可以购买最低配置的,$49/月的...

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