CWD Tuberculosis Found in Spongiform Disease …By 1936, George W. Dunkin reviewed Stockman’s...

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Journal of MPE Molecular Pathological Epidemiology

Lysenko AP PhD1,Broxmeyer L MD2,Vlasenko VV PhD3, Krasochko PA PhD1,Lemish AP1 andKrasnikova EL1

1 InstituteofanExperimentalVeterinaryScience,n.S.N.Wyshelesski,Belarus

2 N.Y.InstituteofMedicalResearchinBayside,NewYork,USA

3 PodolskCenterofTuberculosis,Ukraine

Corresponding author: Dr.LawrenceBroxmeyer,MD

[email protected]

N.Y.InstituteofMedicalResearchinBayside,NewYork,USA.

Tel: 011-718-229-3694

Citation: LysenkoAP,BroxmeyerLMD,VlasenkoVV,etal.CWDTuberculosisFoundinSpongiformDiseaseFormerlyAttributedtoPrions:ItsImplicationtowardsMadCowDisease,ScrapieandAlzheimer’sJMolPathEpidemol.2017,3:2.

CWD Tuberculosis Found in Spongiform Disease Formerly Attributed to Prions: Its

Implication towards Mad Cow Disease, Scrapie and Alzheimer’s

AbstractThe TSE’S or transmissible spongiform encephalopathies, include bovinespongiformencephalopathy(alsocalledBSEor“madcowdisease”),Creutzfeldt–Jakobdisease(CJD)inhumans,and“scrapie”insheeporgoats(caprinespongiformencephalopathy).Theyremainamystery,theircausestillhotlydebated.Currentmadcowdiagnosisliessolelyinthedetectionoflateappearing“prions”,anacronymforhypothesized, geneless,misfoldedproteins, somehowclaimed to cause thedisease.Yet laboratorypreparationsofprionscontainotherthings,whichcouldinclude unidentified bacteria or viruses. And the only real evidence that prionoriginatorStanleyPrusinerhadinhisoriginalpaperthatthediseaseagentbehind“Scrapie” in sheepandgoatswasdevoidofDNAorRNA–wasbasedupon thefactthathecouldn’tfindany.Furthermore,therigorsofprionpurificationalone,might,inandofthemselves,havekilledanycausativemicroorganismandHeinoDringer,whodidpioneerworkontheirnature,candidlypredicts“itwillturnoutthattheprionconceptiswrong.”RoelsandWalravensaswellasHartlytracedMadCowtoMycobacterium bovis.Moreover,epidemiologicmapsoftheoriginsandpeakincidenceofMadCowintheUK,suggestivelymatchthoseofEngland’sareasofhighestbovinetuberculosis,theSouthwest.Theneurotaxicpotentialofbovinetuberculosishasforsometimebeenwellknown.By1911AloisAlzheimercalledattentionto“acharacteristicconditionofthecorticaltissuewhichFischerreferredtoas‘spongycorticalwasting”inAlzheimer’sdisease(AD).ButbehindAD,FischersuspectedamicrobecalledStreptothrixwhichwasconstantlybeingmistakenandconfused for tuberculosis. Our present investigation of the TSEs clearly showscell-wall-deficient(CWD)tubercularmycobacteriapresent,verifiedbymolecularanalysis,ELISA,PCRandmicroscopytocausespongiformencephalopathy.

Keywords: Prions;Scrapie;TheSpongiformEncephalopathies;Alzheimer’sdisease;TheetiologyofAlzheimer’sDisease;Mycobacterium tuberculosisComplex

Received: April05,2017; Accepted: April27,2017; Published: April29,2017

IntroductionWith the transmissible spongiform encephalopathies (TSE’s),including“scrapie”and“madcow”inanimals–mentalandphysicalabilitiesdeteriorateandmyriadtinyholesappear inthebrain’scortexcausingittoappearlikeasponge(hencespongiform).Such"spongiformchange"iscausedbyintracellularvacuolesinneuronsandglia.YetthrougheonsofevolutionandasasurvivalstrategyitisthroughtheformationofjustsuchintracellularvacuolesthatTBandthemycobacteria,unlikemostpathogens,havesurvived,

adopted,andmultipliedeitherinthemacrophagesofthebodyortheneuronsandgliaofthecentralnervoussystem[1-3].Theverysameintracellularvacuolesthateventuallyleadtosponge-likechangesinthebrainforminmicrogliawithinafewhoursofan infection with fowl tuberculosis (Mycobacterium avium) –whichshiftsitsmorphologyfromthickshortmycobacteriatothelongerfilamentousformssooftendocumentedinthespongiformencephalopathies[1].AndasimilarscenarioisoperativeforM. tuberculosisandothermycobacteria,allofwhichthriveinthesevacuoles,possiblycreatingareservoirinthebrainthroughwhichadditionalmicrogliaandneuronscanbeinfected.

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Today,theTSE’sarethoughttobecausedby“prions”characterizedby neuronal loss, gliosis, spongiform change and intracellularvacuoles inneuronsandglia-yettheseverysamechangesareinstigatedbytuberculosisanditsrelatedmycobacteria.MicrogliainternalizesvirulentM. tuberculosismorerapidlyandefficientlythanlessvirulentstrains.Uponinternalization,tuberclebacilliarefoundinsparse,butdenselypackedvacuoles(Figure 1).

Clinicallywhatisseeninallthespongiformencephalopathiesisarapidlyprogressivedementia,andtheirmostcommonclinicalpresentation in humans is CJD or Creutzfeldt–Jakob disease aswell as in certain cases of Alzheimer’s disease. In the opinionof experts, ample justification exists for considering a similarpathogenesisforAlzheimer’s,Creutzfeldt–Jakob,scrapieandtheotherspongiformencephalopathiessuchasMadCowdisease.Infact,Creutzfeldt–JakobandAlzheimer’softencoexistandatthispoint are thought to differmerely by time-dependent physicalchanges.Onestudylinkedupto13%ofallAlzheimer’svictimsasreallyhavingCreutzfeldt-Jakobdisease[4].

In 1909, Sir Stewart Stockman, Chief Veterinary Officer of theRoyalCollegeofVeterinarySurgeons,whileinvestigatingadiseaseofsheepinScotlandknownlocallyas“scrapy”or“scrapie”,foundacid-fast bacilli characteristic of fowl or cattle tuberculosis inlesions of animals dying from scrapie [5]. Stockman will laterbementioned as one of the historical figures in the discoveryof prion disease, but the fact that he found acid-fast bacilli inscrapie,apremierfindingfortuberculosis,wouldnot.

By 1911 Alois Alzheimer noted in some Alzheimer’s cases “acharacteristic conditionof the corticaltissuewhichFischerhasreferredtoas'spongycorticalwasting'."Alzheimerwasreferringtothefindingofhisgreatrival,neuropathologistOskarFischerofPrague.Fischernamedandwasoneofthefirsttodescribesuchspongiformcorticalatrophy[6].ButFischerwasinvestigatingsuchAlzheimer’sbrainchangesinthecontextofbeingfromamicrobecalled Streptothrix, which was constantly being mistaken forandconfusedwithtuberculosis.AndjusttenyearsbeforeOskarFischer found Streptothrix-like forms in Alzheimer’s cerebralplaque, Babèş and immunologist Levaditi reported that onceinoculatedwithtuberclebacillibeneaththeduramateroftheirbrains, rabbitsnotonlydevelopedbranchedfilamentous formssimilar to Streptothrix, but rosettes that were identical to the"drüsen" thatOskarFischer repeatedly reported inAlzheimer’splaque[7].

By 1936, George W. Dunkin reviewed Stockman’s findings,confirming acid-fast organisms in the “scrapie” of sheep andgoats.Butunlikethebovineconditionknownasparatuberculosis(asubspeciesoffowltuberculosisorMycobacterium aviumfoundmainlyincattle),theacid-fastformsofTBconfirmedbyDunkinin“scrapie”werelongerandmoreslenderthenthoseencounteredinparatubercularJohne’sdiseaseincattle.

Notonlyisbovinetuberculosis(Mycobacterium bovisorM. bovis)capableofinfectingallanimalsandmensubjecttothespongiformencephalopathiesbutby1868Jean-BaptisteAugusteChauveauinFranceandEdwinKlebsinPragueproposedthateatinginfectedmeat could transmit bovine tuberculosis [9,10]. SubsequentlyAndreas Gerlach’s experiments confirmed that either milk orfleshfromtubercularcowscouldtransmittuberculosistootheranimals towhich theywere fed [11]. Schliesser also sawmeatfrom tuberculous animals as constituting a significant risk tohumansifavailableforconsumption[12].Withregardtobovinetuberculosis, anyone handling or consuming meat from aninfectedanimalisatriskofcontractingthisdisease.Bytheturnofthe20thcentury25%ofthemanyUSdeathsfromTBinadultswerecausedbyM. bovis.

While various theories continued to swirl around the cause ofTSE’s, the best epidemiologicmaps of the peak incidence andprevalenceofBSEormadcowdisease,doneintheUK,itturnsout, suggestively matched those of the highest prevalence ofEngland’s bovine tuberculosis in cattle, with a predominantdistribution in the Southwest (Figure 2) extending to countiesfurthernorth-theveryareawhereBSEintheUKbegan[13,14].

The prion hypothesisThe TSEs or transmissible spongiform encephalopathies, arecurrently alleged to be caused by “prions.” But the theorysurroundingneurologistStanleyB.Prusiner’s“prion”,awordwhich he himself coined for gene-less proteins that wereinfectious, was under a rightful cloud of suspicion from itsonset[15].

All living organisms, including germs, use their DNA and RNAto make copies of themselves and amplify themselves in thebrainsofthosethey infect.ButprionssupposedlydidnothaveDNAorRNA.Therefore,Prusiner’stheorywasfelttobeheretic,unorthodox,andcontrarytoacceptedbelief.Andtheonlyrealevidencethathehadinhisoriginalpaperwasthatthediseaseagentbehind“scrapie”wasdevoidofDNAorRNA–wasthathecouldn’tfindany.Notonly isscrapiedifficulttodiagnosebut itis always fatal, adegenerativedisease thataffects thenervoussystemsofsheepandgoats.Thename“scrapie”isderivedfromoneoftheclinicalsignsofthecondition,whereinaffectedanimalswillcompulsivelyscrapeofftheirfleecesagainstrocks,trees,orfences.Thediseaseapparentlycausesanitchingsensationintheanimals.ScrapieisthemostcommonreportablediseaseingoatsandsheepintheUnitedStatestoday.

Previous studies in which he had been involved in suggestedto Prusiner that theremay be similarities between the agentscausing scrapie encephalopathy in goats and Creutzfeldt-Jakobdisease (CJD), a rare but fatal disease in humans [16]. YetPrusiner’sinitial1980scrapiepaper,donewithHadlow,Kennedy,

MicrogliainfectedwithM. aviummycobacteriaareinone of the cytoplasmic vacuoles. (Arrow) Bar=5 µmZiehl-Neelsonstain.

Figure 1

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and Race mentions absolutely nothing about prions, referringinstead to “unconventional viruses”, “the CJD virus” and “thescrapievirus”.

All things considered, Prusiner’s idea wasn’t new. The thoughtsof biologists in the 1930s who incorrectly said that viruses wereonlyproteinsandthat‘slowviruses’mightbegene-lesshadbeenproposedbyand discarded inBritainbyGriffithasearlyas1967[17]. Tobe certain, Britishmathematician J.S.Griffithwasfirst toproposenotonlyaself-replicatingproteintheory,butthatproteinscouldbechemicallyidenticalbutsimplyfoldeddifferently.

Thefindingthatprionswereproteinsnormallyfoundinthebody,including the brain of healthy controls, seemed to contradictthebestevidencethattheywereinfectious.Thetheory, likeallquestionablescience,survivedbyfindingadifference:prionsfromhealthy animals were quickly claimed to be “cellular” protein;those from scrapie were “scrapie” protein. Scrapie proteinaggregatedintorodswhilecellularproteindidnot.Anotherso-called “critical clue” [18]: scrapie protein survived proteinase,while “cellular” did not. This still did not mean however thatsomevirusorbacteriaormycobacteriadidnotcausethechangebeing attributed to prions to begin with. Cow tuberculosis(Mycobacterium bovis), for example, both by virtue of its cell-wall-deficient, viral-like forms, and that it sharesmethyllysineswithothermycobacteriaisalsoprotease-resistant[19].Didthismeanthattuberculosiswasalsogeneless?

TheamyloidproteinsinAlzheimer’s,whichasearlyashis1982ScrapiepaperPrusinerwassuggestingbelinkedtoprions,were

alsoknownat thattimetobeproteaseresistant,butnot fromprions[20].

By all logical estimates, the death-knell to the prionhypothesis should have occurred with Lasmézas’s 1997interspecies transmissionofmad cow inwhichmore thanhalfof injectedmicehadnodetectableprions [21]. If thiswasnotenough, then therewasBakerandManuelidis’s2002 studyoninfectiousneurons(microglia)withlowprionlevelsinotherwisehighly infectious material, which only served to support theconceptthatpathologicprionsweretheresultofinfectionratherthanbeingtheactualinfectiousagent[22].

WhenYale’sLauraManuelidisandSotiriosBotsiosrevisitedthissubjectin2016theyoutrightstatedthatCJDandScrapierequireagent-associated nucleic acids for infection, therefore turningtheentireoriginalprionhypothesisonitshead[23].Manuelidiscalled for a re-opening of the entire prion concept to find outjust which genetic material was involved, and its origin. ToManuelidis thiswas likely tobeavirus,althoughsheadmittedthefundamentalmysteryremained.Infact,tomanydissenters,some other, not as yet identified pathogen such as a virus,bacteria, or mycobacteria caused “prions” to misfold — thusdamagingthebrain.

Cell-Wall-Deficient Mycobacteria Longbeforethepriontheorywasproposed,scientistssearchedunsuccessfully for the virus or viruses that might cause TSE,including Dr. Stanley Prusiner. And even today, some speak in

(Left)MadCow-positive(BSE)cattleinUK1997;(Right)BovinetuberculosisinUK1999―bothwithapredominantdistributionintheSouthwest,extendingtocountiesfurthernorth,theveryareaswhereBSEintheUKbegan.

Figure 2

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termsof theTSEs (transmissible spongiformencephalopathies)beingcausedbyactivelyreplicating infectiousparticlesofviral-size and density. But “viral-sized” does not necessarily meanvirus.

Tuberculosisisaninfectiousdiseasethatisfoundthroughouttheworldandwhichcanhaveadramaticimpactontheeconomyandonhumanandanimalhealth.Inhumans,theprincipalcauseofinfectionisMycobacterium tuberculosis,buttheanimal-specificpathogen, M. bovis, is arguably the most important zoonoticagentinhumanhistory[24].

TuberculosisduetoM. bovis hasaworldwidedistribution,andrepresentsoneofthemostimportantpublicandveterinaryhealthproblems[25].Itisofgreatestconcernincattleeventhoughitcanoccurinhumansandmanydomesticatedandwildanimalspeciesincludinggoats,swineandsheep.M. bovis isanimportantfactorinhumantuberculosis[26].Furthermore,inatleastoneformoftuberculosis in sheep and goats― paratuberculosis, the woolorhair isoftendamagedandeasilyshed,notunlike inscrapie.Signsarerarelyevidentuntiltwoormoreyearsaftertheinitialinfection, which usually occurs shortly after birth. And oncesymptoms appear, paratuberculosis is progressive and affectedanimalseventuallydie.

Ofall thebacteria, thepreferredandpredominant formof themycobacteria is their cell-wall-deficient “L” forms which arecrucialtotheirsurvivalinthebody.Anditisherethatdiagnosisbecomes most problematic [27]. Mycobacteria are difficult todetect,especiallyintheirviral-like,obscurestate.Similartoprions,mycobacterialL-formsescapedestructionbythebody’simmunesystem, and are seemingly indestructible. Tubercular L-forms,no matter their exact mycobacterial species, are remarkablydifferent from L-forms of other bacteria in their resistance tochemicalandphysicalagents[28,29].AndthesmallestandmostresistanttosuchenvironmentalstressesaretheirfilterabletinyDNAcontainingL-granules–althoughotherpolymorphicviral-likeandstaph-likecoccoidformsalsoform[30].Yetgeneticanalysisby16S rRNA 16S ribosomal RNA confirms them as tubercular.Despitewhatisreferredtoasprions“heterogeneitywithrespecttosize”―thisisalsoawell-knowncharacteristicofpolymorphicCWDmycobacteria,with theirdiminutive L-granulesmimickingresistant “small proteinaceous particles” in every way. So thefact that CWD tuberculosis is protease resistant or “resistantto inactivation by most procedures that modify nucleic acids”comesasnosurprisewithouttheneedtolabelthem“protease-resistantproteins.”

Itisintherelativelydormantstateofcell-wall-deficiencythatthemycobacteria, not unlike prions, develop enhanced resistanceto harmful factors and a long life– a survival strategy that hasallowedthemycobacteriatobecomeoneofthemostsuccessfulpathogensontheplanet.Butthedownsideisthatsuchdormantcell-wall-deficient or “L-forms” also become among the mostdifficult to cultivate and identify, especially in their early non-cultivable or so-called “invisible” stage [31]. Therefore to findthem in the living organism (in vivo) takes mandatory novelstrategies including special growth techniques to enrich andrevive them to an actively growing, colony-forming state. ToZhang thismeant the resuscitationofdormantMycobacterium

tuberculosis by phospholipids or specific tubercular proteinpeptides[32].However,thesameCWDgrowthstimulationcanoccurwithouttheuseofmycobacterialelements,suchastheuseofgrowthstimulantswhichcreatenutrientstarvationorhypoxicconditionsforM. tuberculosis in vitro[33].Witheithermethod,CWDmycobacteria–otherwisenon-cultivable,will grow,oftentransitioning from ovoid or egg-shaped to rod shaped cells. Infact,therecoveryoftheabilityofmycobacteriatocultivatehastraditionally been associated with the formation of the samerod-shaped, filamentous and granular forms that make CWDtubercular forms indistinguishable fromprions [34].WeusedamycobacterialCell-Wall-Deficientgrowthstimulantwithoutanymycobacterialcomponents.

Amyloid: the common denominator in all Spongiform Encephalopathies“It is an astounding finding, because we never would have dreamed that amyloid and prions are the same”, proclaimedStanley Prusiner [35]. But previous to this and throughout thehistoryofmedicineamyloidwasusuallyconsideredthedepositionthattookplacedueinthecourseofchronicinflammatorydisease(withgenes),mainlytuberculosis-itsusualprecipitatingcause.

Hass’s study at Cornell proved a direct correlation betweenamyloid deposition and the mycobacteria by injecting M. bovis into rabbits and followingM. tuberculosis inhumans.Heconcluded that the only infectious diseasewhich served as anapparentcauseofamyloidosiswastuberculosis[36].Allofthe21humansubjectswithamyloidinHass’sinvestigationhadchronicpulmonarytuberculosis.

Inaddition,ina50-yearstudybaseduponautopsy,Schwartzsawamyloidosis,primaryandsecondary,inthebrainandelsewhereas a by-product of underlying infectious tuberculosis, eitherreactivating itself or being reactivated by a host of traumatic,chemical, biologic or physical insults [37]. Microscopically, inthe brain, Schwartz found plaques and amyloid degenerationof nerve fibrils. When Schwartz injected 22 guinea pigs withM. tuberculosis, all but four came downwith amyloidosis. Hisuninfected controls, with the exception of one, showed noamyloid. He thereby confirmed Hass, whose large series ofrabbits showed that three out of four inoculated with bovinetuberculosishadamyloiddiseasewithin15months[36].Hass’samyloiduniformlyshowedaprincipalproteinfraction.

Theamyloidissuesurfacedagainwhenin1978,ResearcherPatMerz,inbreakthroughwork,identifiedtinyfibrilsinthebrainsofscrapie infectedmicenotpresent inwellcontrols.PrionpuristsrefusedtoadmitthattheirprionrodswererelatedtoMerz’sfind,citingherentitiesaslongerfibrilsandclaimingthatMerzstatedplainlythatherscrapie-associatedfibrils(orSAF)werenotamyloidandthereforecouldnotbeprionrods–thetermPrusinerusedforamyloidfibrils.ActuallyMerzsaidthatherscrapieassociatedfilamentswereamyloid-likeonmorethanoneoccasionandotherworkersinthefieldsuggestedthatthetwoentitiesinMerzandPrusiner’spaperswereidentical[15].Delgadosawsuchfibrilsineithercaseastypicalfeaturesofamyloid[38].

Meanwhile,by1994,deBeer,studyingtherelationshipbetweenamajor rise of serum amyloid and having tuberculosis, saw a

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rapid descent in amyloid in patients treated with anti-tuberculardrugs[39].AsanoffshootofdeBeer’swork,Tomiyamadissolvedβ-amyloidplaquewithrifampin,afirstlinedruginthetreatmentofTB,andoneofthefewagents,tothisday,thatisabletodissolveamyloidplaque[40].

Whether in humans or animals, it is impossible to addresstuberculosiswithoutspeakingofitscell-wall-deficientor“L-forms”which immediately start to form in the body’s macrophage andmicroglialdefendersonceunderattackandcontinuetopropagatethereafter[41-43].

ThepurposeofthisworkwasthereforetoestablishwhethercentralnervoussystemCWDtubercularinfectioncouldresultandaccountfor in theclinicalandhistopathologicalchanges thatweassociatewithtransmissiblespongiformencephalopathies(TSEs).

Materials and methodsTransmissible Spongiform Encephalopathy Fragments from thebrainsof twogoatswithadvanced clinicaldamagetothecentralnervoussystemandclinicalsignsofscrapiewere fixed in 15% formalin (1 month). Laboratory diagnosticsalready performed had ruled-out rabies or listeriosis. Brainfragments,afterbeingwashedwithformalininsterilewaterwerehomogenizedandincubatedonamycobacterialgrowthstimulant(1:4) containing 0.1% chlorhexidine for 48 hours, allowing forthe growth and eventual isolation of cell-wall-deficient (CWD)tubercular/mycobacterialforms[44-46].Thiswasthenplantedintesttubeswithasolidmycobacterialnutrientmediacontainingagar,asparagine,peptone,glutamate,andtheelementsK,Mg,Zn,Mn,FeandSandPandincubatedat37°Cforanadditional10 days. A repeat observation and mycobacterial probe wasconductedafter5yearsofpreservationinformalininBrain1andafter3yearsinthecaseofBrain2.

MicroscopyHistologicalsectionsofthecortexandothersitesofthebrainwerepreparedbystandardtechniqueandstainedwithHematoxylin-Eosin stain, Ziehl-Neelsen, and modified immunoenzymetechnique(MIET)[45,47].MIETstainingprocedureincludedfixingat65°Cfor2hoursfollowedbythesuppressionofendogenousperoxidase with 3% H2O2. Staining by Kinyoun carbol-fuchsinwasdonefor5minutes,followedbydecolorizationwith3%acidalcoholfor70seconds,counterstainedby0.3%methylenebluefor2minutes.FurtherconjugatesofaffinitypurifiedantibodytoM. bovisweredrawnandthenplacedinPhosphate-bufferedsaline(PBS)with0.02%Tween20for1½hoursfollowedbyemersionintoasubstratesolutionconsistingof10mg.3,3'-diaminobenzidine(FlukaSigma-Aldrich) in2mlofDMSOand10mlof deionizedwaterwith10µlof33%H2O2fortwentyminutes.

Ateach stage, smearswerewashedwithdeionizedwaterwith0.02%ofTween20.MIET(modifiedimmunoenzymetechnique)stained slides showed either classical acid-fast (AF) tubercularbacillistained inred,ornon-acid-fast(NAF)cellsorantigenstomycobacteria in brown.Othermicroflora andtissue cellswerestained blue. Results of microscopy were recorded using anOlympus51BXmicroscope.

Cell-Wall-Deficient (CWD) Tubercular reference standards used included isolates fromthe1stInternationalstandardPPDforMycobacterium bovis,isolatesfromthebloodofaTBpatient,isolatesfromthelymphnodeofa tubercular cow, isolates from embryonic (fetal) bovine hearttissue, and CWD forms of M. bovis BCG and M. bovis Vallée.That all CWD isolates were from the genusMycobacteria wasconfirmed both by the existence of common antigens withreferencestrainsofM. bovis,M. avium-paratuberculosiscomplexandM. tuberculosisaswellasPCRswithprimersfor16SrRNA,MPB70–amajorproteinantigenofMycobacterium bovis,andprimersforM. avium-paratuberculosiscomplex.

SDS-PAGE electrophoresis and Western blottingMycobacterial CWD isolates were washed three times with a0.5%phenolsolutionsuspendedinstockbufferandwarmedfor5minutesat99°C.LysatesofisolateswereseparatedbySDS-PAGEelectrophoresisin15%ofPAGE(Laemmli,1970).

Polymerase Chain Reaction (PCR)The identification of isolates was carried out by 16S rRNAmycobacterial primers, MPB70 complex tuberculosis-bovisprimersandprimersforMycobacterium avium-paratuberculosiscomplex(Pramytech).Suspensionsofisolatesinlysingbufferwerewarmedfor5minutesat95°C.TheDNAwasisolatedoncolumnswithasorbent(IBOHNANB)andamplificationwascarriedoutonaC1000TouchTMThermalCycler(BioRad)accordingtostandardprotocols. An electrophoresis of amplificates was then carriedout in 2% agarose gel and results obtained from a MolecularImager®GelDocTMXR+system.(BioRad).

ELISA ELISAofthesonicate isolatesfrombothbrainsweredonewiththe rabbit Antisera to sonicates of M. tuberculosis H37Rv, M. bovis ValleeandM. avium1603.

ResultsSpongiform changes found in tubercular cortex Multiple brain thin cuts were performed at the level of thecerebralcortexandtheponsintwotuberculargoatswhichbeforedyingdemonstratedtheclinicalsignsofadvancedtransmissiblespongiform encephalopathy (scrapie). Spongiform changescaused by intracellular vacuoles, accompanied by thromboticvessels,proliferationofcerebralvessels,perivascularcuffing,andlymphocyteinfiltrationwereallinevidence.Inadditiontoporousspongiform changes there was also a proliferation of heavilyvacuolatedglia(Figure 3).

Cell-Wall-Deficient tubercular formsAt microscopy, some of the smears of a brain stained withZiehl–Neelsen (ZN) stain yielded various cell-wall-deficientmycobacterial forms. Among these were partially acid-fastsphericalshapes(Figure 4a-4c)whichweregreaterinnumberinBrain1,andrarerinBrain2.Inonesuchcase,apartiallyacid-fastsphere(spheroplast)isactuallyseenconnectedtoapartiallyacid-

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Thinhistologicslicesfrombothbrains.(a)fromBrain1.(b)isfromBrain2.Sponginessinbothbrainsnoted.Haematoxylin-Eosin,10X100.

Figure 3

bа

C

(a) Inbrain1, therewere redspherical shapes (blackarrows).Also (redarrow),a red rodwasconnectedwithoneof thespheres,Z-N,10×100;(b)Isfrombrain2,withasimilarsphericalshapeZ-N,10×100;(c)Frombrain1,showsbrown(non-acid-fast)mycobacterialsphericalshapesandared-brown(partiallyacid-fast)rod,MIET,10×100.

Figure 4

fast(redcolor)rod(pinkarrowinFigure 4a).MIETstainedthesespheroplastsbrown,indicatingnon-acid-fast(NAF)mycobacterialforms(Figure 4c).AlsoseeninFigure 4cisoneoftheoccasionalacid-fastrodsseen(Figure 4c).

MIETstainingofbrainspecimensalsorevealedbrownrods(Figure 5).SuchrodsweremoreprevalentinBrain2.Therodsfoundinbothbrainshadalmostidenticalmorphology(Figure 5);aswellashavingadoublerodconfigurationofsimilarmorphology.Doublerods with similar morphology were also found in tubercular

embryonic(fetal)cardiacsmearsobtainedfromatubercularcow(Figure 5c).

AntibodiestoM. bovisreactedwithautopsiedtissuefrombothanimalswithadvancedscrapie,makingpossiblethevisualizationof other commonly encountered tubercular cell-wall-deficient(CWD) forms and mycobacterial antigens. In Figure 6a, thewhitearrowpointstoaneuronwhilevariousCWDnon-acidfastmycobacterialinclusions(brown)inthecytoplasmcanbeseeninFigures 6a-6e.Suchbrown inclusions in thecytoplasm (Figures

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6a-6e) are often surrounded with a brown web-like syncytialnetwork (6e).Figure 6b showsbrownfibrils similar to scrapie-associated-fibrils(SAF).

Fragments of brain tissue were washed in sterile water andhomogenized.UponPCR,primers forboth16sRNAandbovinetubercularMPB70gavepositivereactions(Figures 7).

Therodsofbovinetuberculosisareoftenspindle-shaped.Afterrepeatedsubcultures,thecolorofthecellsmaintainedagreenshade that is connectedwith theweak synthesis of tubercularantigens. Isolates from both scrapie brains were polymorphic,yet shared common forms between themselves and CWDtubercular isolates from other sources. In smears, it was notunusual to see CWD mycobacterial forms in the same fieldthatwere longandshortbipolarrods, thingranularrods, thick"empty"rods,andspindle-shapedforms(Figure 8).Thesesameforms were found also in CWD isolates from tuberculin PPD[44], as well as from humans and cows sick with tuberculosis[44].Verycharacteristically,asseeninFigure 8,aretheformsoftworodsseeminglyboundtogether,withaseptuminbetween– a configuration found in isolates from our brain segments,thetuberculins,andthebloodofatubercularpatient,possiblyrepresentingaprocessofdivision.Suchtubercularseptationcancreatetwodaughtercells[48,49].Again,therodsofthebovinetubercularbacillusareoftenspindle-shaped(Figure 8).

WithSDS-PAGEelectrophoresis,bothbrainisolateshadidenticalpolypeptideprofiles(Figure 9,lane1and2)whichwere,inturn,almostidenticaltoboththeprofileforPPDtuberculinPPD(Lane3)andthatofaCWDM. bovisisolate(Lane4).

In addition, Western blot revealed three amino acid (40-70kDa)fractionsfrombrainisolatesreactedwithantiserumtoM. bovis,aswellassimilar fractionsof isolatesfromstandardPPDtuberculinofM. bovis (Figure 10). Similar triple fractionswerealsofoundinatypicalMTBsonicate.

UponELISA,isolatesfrombothbrainsreactedtotheantiserumsof M. tuberculosis, M. bovis and to a lesser extent M. avium

(Figures 11 and 12).

The subsequent formalin-resistance of the CWD tubercularforms taken from our brain specimens, even after 3 to 5 yearexposure to 10% formalin were of particular interest. Brainsamples (3-4Gms) taken from formalin solutions under sterileconditionswere placed in 100ml of sterilewater. Thesewerethenhomogenizedin100mlofsterilewater.After12hoursthesewere again homogenized with 10 ml of mycobacterial growthstimulant,incubatedfor48hat37°C,anda300µlaliquotofthiswasthenthenplantedintesttubescontainingslantedsemisolidmycobacterialgrowthmedium.Thesetesttubeswereincubatedat37°Candaftertwodays,smearsfrombothspecimensbegantoyieldgrowthbutvisiblecolonieswerenotpresent.Butunderblind subculture at 24 hour intervals both series (Brain 1 andBrain2)yieldedsmall,transparent,mergingcolonies,thesmearsofwhichshowedthesamepolymorphicrhabdoidspindle-shapedforms(Figure 13a)designatingbovinetuberculosisasoriginallyseen.Anddespitethelargetimeintervalof3to5yearsseparatingtheseassaysfromthoseoftheoriginal,theystillreactedwiththeantibodyofM. bovis,andthesamemycobacterialPCRprimers(Figure 14)andgavethecharacteristicbrown-colorfornon-acid-fastbacilliwithMIETstain.

DiscussionSpongiformandotherpathologicalchangessimilartoBSE(MadCow Disease) were found in goats with the clinical symptomsof scrapie, who fell ill with signs of a central nervous systemlesionintheirbrain.Yetinassaysofbothexperimentalanimal’sbrains, partially acid-fast tubercular cell-wall-deficient (CWD)spheroplasts,rods,andothermycobacterialCWDforms―alongwith theexistenceof common tubercular antigenswere found[50]. Other known infectious diseases yielding similar brainlesionswereexcluded.

Besidesthis,net-likestructuresreactedwiththeantibodytoM. bovis whichwereeithertheresultoftubercularantigensoractualCWD tubercular forms developed a net-like syncytial structure

a b

c (a)–frombrain1,(b)frombrain2,(c)embryonic(fetal)cardiactissuefromatubercularcow.Brown“double"rods,ofidenticalmorphology,allfields.MIET,10×100.

Figure 5

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a

c d

e

b

(a) is frombrain1.The longbrownstructure(whitearrow) isaneuron,whiletheblackarrowpointstotwonon-acid fastbrownspheroplasts.AlsopresentaretwolargeCWDmycobacterialformswithdarkbrowncytoplasm(bluearrows).(b)showsscrapieassociatedfibrilsinbrain1,whichalsoprovedtobetubercularonmolecularanalysis.(c)FromBrain2showsbrowntubercularCWDformsinfiltratingtwocellsaswellastwobrownrods(blackarrows).(d)FromBrain2showsthesamebrownNAFtubercularinclusions.AlsofromBrain2,(e)shows2largecellssurroundedbyaweb-likesyncytialstructure,againshowingsignsofmycobacterialinfiltration.MIET,10×100.

Figure 6

PCRofDNAfromhomogenatesofBrain1(1)andBrain2(2).k–Negativecontrol,k+positivecontrol.Figure 7

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similartoscrapieassociatedfibrils (SAF).Yetonthegroundsofmorphology, partial acid-fastness, the existence of antigens incommonwithMTB,andPCR/ELISA results, itwasobvious thatagain, we were dealing with cell-wall-deficient and classicaltubercular forms. Furthermore, our results simply confirmedstudiesdoneat the turnof the20thCentury,firstbyStockmanand later by Dunkin that spongiform changes in the brains of

animalswithscrapiewerecausedbyacid-fastorpartiallyacid-fasttubercularbacilli[5,8].

Natural transmission of scrapie in the field seems to occur viathe alimentary tract in themajority of cases, and scrapie-freesheepflockscanbecomeinfectedonpastureswhereoutbreaksof scrapiehasbeenobservedbefore.Thesefindingspoint toasustainedcontagionintheenvironment,notablyinthesoil[51].Detectable“infectious”prions(PrPSC)havebeenreportedinthefecesofsheepbothintheterminalandtheearlypreclinicalstagesofthedisease,suggestingtheprionswerelikelytobeshedinto

a b c

(a) An isolate froma Brain 1 showing a partiallyAF rod (red-brown color) shared with formationof a NAF rod (brown color). Cells with similarmorphology are found in isolates from PPDtuberculin(b),andfromthebloodofaTBpatient(c) black arrows. (b) shows the formation of arhabdoidbipolarrodformingaspindle-shapedcell(whitearrow).

Figure 8

SDS-PAGE electrophoresis 15% andwestern blotwithbovineantiserumtoM. bovis(1)IsolatefromBrain 1, (2) Isolate from the brain 2. (3) IsolatefromPPDtuberculin.

Figure 10

SDS-PAGE electrophoresis 15%: (1) Isolatefrom Brain 1. (2) Isolate from Brain 2 (3)IsolatefromthePPDtuberculin,(4)CWDM. bovisBCG.

Figure 9

ELISA of sonicates of the isolates from brain 1with antiserum to M. tuberculosis H37Rv, M. bovisVallée,andtoM. avium1603.Abscissaaxis-dilutionsofantisera,ordinateaxis-OD450nm.

Figure 11

ELISA of sonicates of the isolates from brain 2with antiserum to M. tuberculosis H37Rv, M. bovisVallée,andtoM. avium1603.Abscissaaxis-dilutionsofantisera,ordinateaxis-OD450nm.

Figure 12

a

b

(a) GrowthofseedsofBrain1,(b)Growthofseedsofbrain2.Longandshortbipolarrods,sometimeswithgranuoles(blackarrows),thick"empty"rods(blue arrows), In (a) a spindle-shaped form (redarrow).MIET,10×100.

Figure 13

https://en.wikipedia.org/wiki/Transmission_%28medicine%29

https://en.wikipedia.org/wiki/Feces

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theenvironmentthroughoutthecourseofthedisease.Butthereistheprecisesameabilityfortransmissionwithtuberculosisandthe mycobacteria through the alimentary tract as well as thesustained contagiousness of the Mycobacterium tuberculosis complex andrelatedmycobacteriainthesoil[52].

Thelengthoftimeittakesforsheeporgoatstodevelopscrapieisusually18to24months,aslowcoursewhichisnotunusualintheprogressionofCWDtuberculosis.By1996, Insanovwarnedthataninfectionwithcell-wall-deficientformsofTBinchildrennot onlymade standard treatment less effectivebut created adiseasewithagradual,insidiousonsetandaslowaccumulationof cerebral damage [53]. This made CWD tuberculosis moredifficulttodiagnose,becauseitsslowburnintonervoussystems,allowed months to years before its full spectrum of damagebecameobvious.YetinthissamestudyInsanovalsoshowedthatcell-wall-deficientformsintuberculosismeningitisaccountedforanincredible87.6%ofthetuberculosisfoundinchildrenwithTBmeningitisand87.3%ofcasesofTBmeningitisinadults.

Theoriginalcauseofcerebraltuberculosis,aswellasotherformsofthedisease,canbeasaresultofexogenousinfectionorbecauseoftheactivationofalatenttuberculousinfectionthatcrossedtheplacenta.We foundevidence for thisplacental crosswhen thesametubercular rhabdoid, rod-like forms inourscrapievictimsalso appeared in the embryonic fetal tissue from a tubercularpregnant cow (Figure 5). In addition the lack of characteristiclungandlymphnodeinvolvementinoursubjectanimalspointedtowardsamoresystemictrans-placentalinfection.

Onceapathogen like tuberculosishas its cellwalldisrupted, itbecomeplastic,havingtheabilitytoassumemanyforms.Someare sotiny that theypass througha22-μmfilter, the so-called“viralstage”ofabacteriaormycobacteria.ItisknownthatCWDTBcanformvariousforms,includingthosewithasizelessthan0.22μm(millimicrons)[33,54].BecauseofthisCell-Wall-DeficientformsofMTBcanpenetratenotonlytheplacentabuttheblood-brainbarrier, andget into thebrain.Calmettenoted thatafter

such infection with cell-wall-deficient tuberculosis, quick fetaldeath could occur. But even if the childwere born alive, withnoapparentill-effects[55],theirdiseasealmostinvisible–suchstealth CWD forms of their dormant tuberculosis could springbackatanytimetothediseasesvirulentforms.

Coming as no surprise, a similar and parallel failure to detect“prions”intheuterineandgestationaltissuesfromagravidawithCreutzfeld-JakobDiseasehasbeenreported(Figure 15) [56].

Brieger,workingatCambridgeUniversity,demonstratedmainlypleomorphic stealth growth when he inoculated tuberculosisdirectlyintotheamnioticsacoflabanimals[57].Theouterlayerof the amniotic sac is part of the placenta. Rapidly, cell-wall-deficientgranulesformedthatdidnotstainwithtraditionalacid-faststain.Withinthreedays,othercell-wall-deficientformssuchasthelongbranchingfungalfilamentsofTBappeared―easilymistakenforso-called“ScrapieAssociatedFibrils”(SAF).Briegerrepeatedthestudyusingfowltuberculosis inchickenembryos.Hisresultsweresimilar[58].Cell-wall-deficientformsappeared,againnotidentifiablebytraditionalstainsorcultures.

Tubercular cell-wall-deficient forms, such as we isolated inscrapieareeasilyasresistantandlonglastingasprions.Yettheirproperisolationrequiresspecialmycobacterialgrowthstimulantsandnutrientmediums[44-46].Themechanismfortheviabilityof tubercular CWD forms, even in the face of otherwise lethalthermalandchemicalassaultisamatterofactiveresearch,butjust how this happens is still as yet undetermined. Our brainsamples,whichyieldedsuchformseasily,withstood3to5yearsofstoragein10%formalin,comparabletothevauntedviabilityofprions.Theformalinandhighheatissuesarenosmallpartinthehistoryofprions.Prusinercitedhispredecessor,UKveterinarianLain Pattison throughout his works saying at one point thatPattisonwasoneofthefirsttosuggestthatthe“scrapieagent”mightbeabasicprotein“basedontheresultsofhisattemptstopurify the infectiouspathogen” [59].Actuallyduring the1960sPattison, even before mathematician Griffith, was astonishedbythescrapieagentssurprisinglyhighresistancetoformalinaswellashighheat[60]–propertieswhichheandhiscollaborator

PCRwithDNAofisolatesfrombrain.(16+)positivecontrol forprimers16sRNA, (161) fromBrain1(growth ina test tubeNo.1), (165) fromaBrain2 (growth in a test tubeNo. 5), (70+) positivecontrolforprimersofMPB70,(701)-isolatefromBrain1 (growth inatest tubeNo.1), (705) fromBrain2(growthinatesttubeNo.2).

Figure 14

The emergenceof a typical, familiar classical tu-bercular rods (arrows) from STEALTH pleomor-phic (multi-shaped) cell-wall-deficient tuberculargrowth[50].

Figure 15

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KatherineJoneswerecertainthatwerecloselysimilartothoseof ‘encephalitogenic factor’, which Pattison believed to be‘a basic protein’ [61]. But in reality, studies performed sincePattisonsuggestthatformalinalsodoesnoteffectivelyinactivateMycobacterium tuberculosisorrelatedmycobacteriainformalin-fixed tissue [62,63] – certainly reflected by the fact that ourbrainsamplescontinuedtoyieldCWDtubercularforms–easilywithstanding3to5yearsofstoragein10%formalin.

Aslifeexpectancyincreases,withwaningimmunity,sotoodoesthe probability of acquiring an active TB infection of the CNSand elsewhere. Therefore Alzheimer’s most comprehensive1911 paper was entitled: “On certain peculiar diseases of old age” [64]. Although Alzheimer’s disease, like Parkinson’sdisease, is characterized by extracellular Aß plaque formationand intraneuronal tau protein inclusions – there is significanthesitancytousetheterm“prion”todescribetheiroccurrence.This is primarily because in contrast to CJD and Kuru, thetransmission of Alzheimer’s and for that matter Parkinson’shas not been demonstrated between individuals and mostexperimental prion work on this subject have used transgenicanimalsthatoverexpressdiseaseprotein[65].Transgenicmice,whichhaveaforeigngenedeliberatelyinsertedintheirgenome,overexpress amyloid beta protein and are an incomplete andlimitedmodel forAlzheimer’s disease [66].On theother handtuberculosisandinparticularitsCWDformswereanintegralpartin conceptions regarding Alzheimer’s from its inception to thepresent.By2013MawandaandWallacepointed to twoprimesuspects for Alzheimer’s amyloid-beta deposition: “especially

chronicinfectionsliketuberculosisandleprosy”[67].

Recently, Alteri showed that during active infection, M. tuberculosisproducesouterfringelikebacterialamyloidstructurescalled“fimbriae”or“pilli”onitssurfacethatalsobindCongoreddye,apropertyassociatedwithhumanamyloidosis[68].JordalreconfirmedthatmycobacterialikeTBproducealevelofamyloidnot previously described for any other bacteria. Not only wasamyloidfoundpresentintubercularmycobacterialbiofilms,butintheactualcellenvelopsofthemycobacteria,aswellascoatingthesporestheyproduce[69].

There is no known disease which better fits into what isoccurringinMadCow,scrapie andtherestofthespongiformencephalopathies – including its human counterparts than theMycobacteria tuberculosis complex and its blood-brain barrierpenetrating, viral-like, cell-wall-deficient forms. It is for thesereasonsthatfutureresearchneedstobeaimedinthisdirection.

Acknowledgments Including Sources of Support Therewerenosourcesofcommercialfundingorgrantsforthisstudy.

Conflict of InterestTheauthorshavenoconflictof interest,financialorotherwisewithregardtothisstudy.Proceduresinvolvingexperimentationonanimalsubjectsweredonehumanelyandinaccordwitheithertheguideoftheinstitutioninwhichtheexperimentsweredone.

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