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From: TSS ()
Subject: Spiroplasma as a Candidate Agent for the Transmissible Spongiform Encephalopathies
Date: October 13, 2005 at 11:07 am PST

J Neuropathol Exp Neurol

Cop Copyright yright ! 2005 by the American Association of Neuropatholo Neuropathologists, gists, Inc.

Vol. ol. 64, No. 10

October 2005

pp. 833–838


Spiroplasma as a Candidate Agent for the Transmissible

Spongiform Encephalopathies

Fran Frank O. Bastian, MD


The reco recover ery of a no novel el Spir Spiroplasma oplasma sp. from brain tissues from

sheep with scrapie, cer cervids vids with chronic wasting asting disease, and from

patients with Creutzfeldt-Jakob disease through passage through

embr embryonated onated eggs has raised the issue of the role of Spiroplasma in

the transmissibl transmissible spongifor spongiform encephalopathies (TSE). In this re revie view,

we ha have inser inserted ted into an epidemiolo epidemiologic gic infection model evidence vidence ac- accum

umula late ted over th the pa past st 30 year ars sh showi wing ng in invol olvem ement nt of Spi piropl plasm sma

infect infection ion in TSE. SE. These hese dat data suppor pport our ur hypothes pothesis is that a Sp Spir irop opla lasma sp.

is the causal agent of TSE, although Koch’ och’s postulates must be ful-

filled to definiti definitivel vely ans answer er that question.

Key Words: ords: Chronic wasting asting disease, Creutzfeldt-Jakob disease,

Koch’ ch’s po postulates, stulates, Prion, rion, Scrap Scrapie, e, Spiro Spiroplasma, lasma, Tran ansmissib smissible

spongifor spongiform encephalopath encephalopathy.


Spiroplasma infection has been sho shown wn to be associated

with the transmissibl transmissible spongifor spongiform encephalopathies (TSEs)

(1–6). We ha have ve no now isolated a Spir Spiroplasma oplasma sp. from all for forms ms

of TSE, including scrapie in sheep, chronic wasting asting disease

(CWD) in cer cervids, vids, and patients with Creutzfeldt-Jakob disease

(CJD) (7), using a well-established ell-method in invo volving lving passage

through embr embryon yonated ated chicken egg eggs (8). Ongoing experimen experiments ts

are intent on fulfilling Koch’ och’s postulates (T Table able). ). In this re revie view,

we ha have ve outlined the possible role of Spiroplasma in the

patho pathogenesis genesis of TSE by inser inserting ting data regarding Spiroplasma

into an epidemiolo epidemiologic gic model for TSE infection (9). The

proposed chain of infection model for Spiroplasma in TSE

consists of characteristics of agent, reser reservoi voir of infection,

por portal tal of exi exit from reser reservoir voir, mode of transmission, por portal tal of

entr entry into host, susceptibility of host, and patho pathogenic genic mech- mechanisms

anisms in invol volved ved in infection.


Spiroplasma are a di diverse verse group roup of wall-less all-bacteria

(10, 11) sho showing wing variable ariable mor morpholo phology gy (Fi Fig. g. 1) and gro rowth wth

characteristics (12). These fastidious microbes require media

of high osmolality for gro rowth wth (13). A filterable infectious

agent in the range of 35 nm is kno known wn to cause TSE infection

(14). We ha have ve found Spiroplasma at 40-nm size at all stages of

gro rowth wth cycl cycle in broth culture (Fi Fig. g. 2). Se Severa veral Spiroplasma

species are abl able to induce persistent brain infection in small

mammals (15), the best kno known wn being S. mirum mirum. Clark de- described

scribed suckling mouse cataract agent (SMCA) infection in

neonatal mice after inoculation of a rabbit tick isolate of this

Spiroplasma species (16). Initiall Initially, SMCA SMCAwas as not reco recognized gnized

as a bacterial infection, but subsequent studies defined the role

of Spiroplasma in causing experimen experimental tal persistent vacuolar acuolar

en ence ceph phal alop opat athy in th the mi mice ce (1 17). 7). There is variable ariable vir virulence ulence

for dif different ferent Spiroplasma species in exper experimental imental mamma- mammalian

infection (18). ). SMCA is less vir virulent, ulent, whereas GT GT-48

-Spiroplasma is the most lethal of the S. mirum strains. A

dif different ferent serolo serologic gic group roup of Spiroplasmas represented by S.

mellifer melliferum um produces persistent brain infection in mice but

without patholo pathologic gic changes (19).

Experimental GT GT-48 -Spiroplasma infection in the rodent

induces a spongifor spongiform encephalopath encephalopathy (20). There is a clear

Spiroplasma dose response cur curve ve with high dose being lethal

in 3 weeks, ks, whereas hereas lo low-dose w-inoculum noculum results esults in persistent

Spiroplasma infection in the rodent brain. It is note notewor orth thy

that there are remarkab remarkable le similarities betw between een exp experimental erimental

Spiroplasma infection in rats and TSE. Lo Low-dose w-infection

with GT GT-48 -Spiroplasma produces perineuronal vacuoles, acuoles, lack

of inflammation, and scattered cattered micro icroglia glia (F Fig. ig. 3). ). Golgi

studies sho show bulbous dendritic sw swelling elling and loss of dendritic

spines in both exp experimental erimental Spiroplasma infection and CJD

(data not sho shown). wn). Transmission ransmission electron microscopic studies

of exp experimental erimental Spiroplasma infection in the rat are essentiall essentially

identical to TSE (F Fig. ig. 4).

The concept of a bacterial infection in TSE surel surely fits

with the ability of the TSE agent to adapt or mutate to a more

vir virulent ulent for form (21). It is note notewor orth thy that the ne new isolates from

TSE gro row sluggishl sluggishly in broth culture and ma may lend themselves

to long incubation times (7). The beha behavior vior of the isolate in vi vivo vo

must await ait animal inoculation studies.

A key feature of TSE is lack of an immune response

(22), although there is micro microglia glia proliferation in the CJD brain

(23). Lack of immuno immunogenicity genicity is also a feature of expe experimental rimental

Spiroplasma infection in the rat in wh which ich there is exten extensi sive ve

spongifor spongiform deg degeneration eneration without inflammator inflammatory response (20).

The ability of Spiroplasma strains to sur survi vive ve in the hemol hemolymph ymph

of insects suggests that the host is extremel extremely tolerant to this

microbial infection (24). An explanation ma may relate the near

Fr From om the Depar Department tment of Patholo Pathology gy, Tulane ulane Health Science Center Center, Ne New

Orleans, Louisiana.

Se Send nd cor orre resp spon onde denc nce an and re repr print nt re requ quest sts to to: Dr Dr. Fra Frank nk O. Bas asti tian an,

Depar Department tment of Patholo Pathology gy, Tulane ulane Health Science Center Center, 1430 Tulane ulane

Avenue, venue, Ne New Orleans, LA 70112; E-mail:

Funding for this project has been pro provided vided by National Institutes of Health

gr grant ant # R01-NS044000.

J Neuropathol Exp Neurol ! Volume 64, Number 10, October 2005 833

JOBNAME: jnen 64#10 2005 PAGE: 1 OUTPUT: Tue September 20 15:15:05 2005


100% meth methylation ylation of the CpG sequences of the Spiroplasma

DN DNA (25) so that the bacterium can avoid void the toll receptor and

innate immune response ordinaril ordinarily associated with bacterial

infections (26). There is nor normal mal cell-mediated immunity in

TSE hosts (27).

The unique feature of the transmissible agents of TSE is

the extr extreme eme resistance to heat, fixati fixatives, ves, and radiation (28, 29),

although there has been some debate regar regarding ding the de degree ree of

this resistance (30, 31). We ha have ve sho shown wn that Spiroplasma resist

ex exposure posure to temperatures near boiling (unpub unpublished lished obser observa- ations).

tions). These ex experiments periments were ere carefull carefully conducted with oil

resulting in a unifor uniform heating. We also placed a Spiroplasma

broth culture adjacent to a cesium source for 2 weeks eeks without

ef effect fect on viability viability. We ha have ve also obser observed ved Spiroplasma sw swim- immin

ing in 50 50%gl glut utar aral alde dehyd yde, e, pr pres esum umab ably be beca caus use th the ba bact cter eriu ium

is pr prot otec ecte ted by a li lipi pid po polys ysac acch char arid ide co coat at in inst stea ead of a ce cell wal all. l.

Others ha have ve noted a unique resistance of Spiroplasma to fix- fixati

atives ves (32). The biolo biologic gic proper properties ties of the ne new isolates from

TSE ha have ve not been evaluated. aluated. An interesting phenomenon is

the susceptibility of scrapie agent to tetrac tetracycline ycline as sho shown wn by

inhibition of infection when hen gi given ven with the inoculum (33, 34).

Spiroplasma are resistant to all antibiotics except tetracycline

(unpub unpublished lished obser observations), ations), suggesting a corollar corollary with the

TSE data.


Cur Currentl rently, there is no way to evaluate aluate the number of

TSE-infected hosts because no preclinical screening test for

TSE is cur currentl rently availabl ailable. e. The suggestion of the spontaneous

nature of the occur occurrence rence of TSE is not acceptable (35) because

there is clearl clearly a transmissibl transmissible agent and there is an absence of

TSE infection in geneticall genetically identical animal populations (36).

The source of infection in iatro iatrogenic genic cases is clearcut (37), but

other en environmental vironmental sources of infection ha have ve not been deter- determined.

mined. Bo Bovine vine spongifor spongiform encephalopath encephalopathy (BSE)-infected

animal tissues are highl highly suspect as a transmission source, thus

the concer concern for occur occurrence rence of BSE in our cattle population

(38). The reser reservoir voir for Spiroplasma species kno known wn to produce

exper perimen mental al br brain ain infec ection tion in mammals ammals is not known.

TABLE. Koch’s Postulates

1. A specific microbe must be present in all disease cases.

2. Microbe must be culti cultivated ated outside host in a pure culture.

3. When pure culture of microbe is inoculated into health healthy hosts, disease

symptoms identical to those of the initial host must be reproduced.

4. Microbe can be isolated again in pure culture from this exp experimentall erimentally

inoculated host.

FIGURE 1. GT-48 strain of Spiroplasma mirum seen on negative

stain electron microscopy of log phase broth culture shows

complex budding of filaments from a central core. When

stressed, the Spiroplasma lose the filaments and only the core is

seen in the culture. Original magnification: 50,000 50,0003.

FIGURE 2. Pelleted GT-48 Spiroplasma log phase culture

examined by transmission electron microscopy shows a collec- collection

tion of small bacterial forms measuring only 40 nm in diameter.

These small forms of Spiroplasma are seen at all stages of the

growth cycle and are in the size range of the filterable trans- transmissible

missible spongiform encephalopathy agents. Original magni-

fication: 50,000 50,0003.

834 q 2005 American Association of Neur Neuropatholo opathologists, gists, Inc.

Bastian J Neuropathol Exp Neurol ! Volume 64, Number 10, October 2005

JOBNAME: jnen 64#10 2005 PAGE: 2 OUTPUT: Tue September 20 15:15:05 2005


Spiroplasma ha have ve been isolated from ticks (17) so that the

reser reservo voir ir could be either another animal species or plant.

When spread from ingestion or inoculation of animal

tissues, exp experiments eriments with scrapie ha have ve sho shown wn infecti infectivity vity to

be present initiall initially in the reticuloendothelial system with

event ventual ual localization to the brain, spinal cord cord, and ey eye (39).

This distribution of infecti infectivity vity is the same for experimental

Spiroplasma infection in the suckling mouse (16). A transient

hemato hematogenous genous phase is seen in experimental SMCA infection

(16) that simulates that associated with TSE infection (40),

although there is much debate as to ho how long TSE infection

remains in the bl blood ood (41). TSE has been transmitted through

bl blood ood transfusion (42). Spiroplasma are neurotropic, even ventu- tuall

ally localizing to the brain (43). Ho However ver, these microbes

thri thrive ve on sterols (44), which hich ma may account for lipid-rich

adrenals and ovaries aries being in involv volved ed in experimen experimental tal scrapie

infection (45).


In TSE, there is extensi extensive ve evidence vidence of oral spread (46).

In sheep with scrapie, the lymphoid ymphoid tissue along the

gastrointestinal tract is infectious (47). In humans, cannibalism

accounted for spread of kur kuru (48). Ingesting antacids leads to

increased infecti infectivity vity by TSE tissues, suggesting some sus- susceptibility

ceptibility of the agent to acids (49). Spiroplasma sho show wide

resistance to a wide range in pH (50), probabl probably again attributed

to its lipid pol polysaccharide ysaccharide coat.

An interesting phenomenon is the spread of CWD from

animal to animal and to animals man many yea years rs after a field was as

laid fallo fallow (51). Clearl Clearly, something in the soil was as causing this

infecti infectivity vity but the mode of transmission is unkno unknown. wn. One

possibility is the transmission of Spiroplasma from the soil

car carried ried by a parasite kno known wn to be pre prevalent alent in the endemic

are areas. s. Spirop Spiroplasma lasma are re also so prese present in hemato ematopha phagus gus

insects such as the deerfl fly (52), ), which hich could pro provide vide a

means of spread. It is note notewor orth thy that scrapie has been spread

to other sheep through ha hay mites (53), suggesting another

mechanism that could be exp explained lained by a resident Spiroplasma



Spiroplasma are intracellular bacteria (20) and could

enter the cell through the lipid rafts on the cell surface, thereb thereby

avoiding voiding the immune system (54, 55). The cells lining the

gastrointestinal tract would ould be the initial site of infection

follo followed ed by spread in the local lymphatics ymphatics and event ventuall ually to

the brain (56, 57).

The initiation of infection brings up the question of

the role of the prion in the infection. The prion is an anoma- anomalous

lous folded host protein that is attached to the membrane

FIGURE 3. Histologic section of brain cortex from rat experi- experimentally

mentally infected with GT-48 Spiroplasma sampled 25 days

after inoculation shows spongiform degeneration of the

neuropil with prominent perineuronal vacuoles (long arrows)

and focal microglial proliferation (short arrows). These tissues

contained 10 10-3 -Spiroplasma per gram of brain tissue as assayed

in SP-4 broth. Hematoxylin and eosin staining; original magni-

fication: 400 4003.

FIGURE 4. Electron micrograph of Spiroplasma-infected rat

brain depicted in Figure 3 shows spongiform degeneration of

the neuropil remarkably similar to the neuropathology of trans- transmissible

missible spongiform encephalopathy brains. Although Spiro- Spiropl

plas asma ma wer ere cu cult ltur ured ed fro rom th the rat at br brain in ti tiss ssue ues, s, no Spi pirop opla lasm sma

we were id iden enti tifi fied ed in th the sa samp mple le by el elec ectr tron on mi micr cros osco copy py. Ori rigi gina nal

ma magn gnifi ifica catio ion: n: 15 15,00 0003. Re Repr prin inte ted fro rom The he Am J Pa Path thol ol 19 1984 84;

11 114: 4:49 496– 651 514 wit ith pe permi miss ssio ion fro rom th the Ame meri rica can Soc ocie iety ty fo for

Inv nves estig igati tive Pa Patho holo logy gy.

q 2005 American Association of Neur Neuropatholo opathologists, gists, Inc. 835

J Neuropathol Exp Neurol ! Volume 64, Number 10, October 2005 Spiroplasma in TSEs

JOBNAME: jnen 64#10 2005 PAGE: 3 OUTPUT: Tue September 20 15:15:08 2005


Fig. 3 li live ve 4/C

surface (58). The abnor abnormal mal prion isofor isoform misfolds within the

cy cytoplasm toplasm of the cell and event ventuall ually is deposited in the

lysosome ysosome (59). A recent paper by Watarai atarai et al has sho shown wn that

the nor normal mal prion on the membrane surface ser serves ves as a receptor

protein for initiation of a bacterial infection, Brucella abor abortus tus

(60). If we assume that Spiroplasma similarl similarly use the prion as

a receptor protein, this mechanism would ould expl explain ain a possible

relationship to prion. Presumabl Presumably, like with Brucella Brucella, Hsp60

on the surface of the bacterium binds to the prion and ini- initiates

tiates inter internalization nalization of the bacterial Hsp60 prion complex

into the cell at the site of lipid rafts. Prion has been sho shown wn

to bind to members of the Hsp60 chaperone famil family (61, 62).

The cyc cycling ling of nor normal mal prion isofor isoform into the cell is kno known wn

to occur at the site of membrane rafts (63, 64). Studies are

underw underway to confir confirm this hypothesis ypothesis and to deter determine mine if mis- misfolded

folded prion protein is produced in the course of ex experimen- perimental

tal Spiroplasma infection in the rodent. At the time of our

prior study (20), monoclonal antibodies against prion protein

were ere not available, ailable, so, although we ha have ve demonstrated that

scrap scrapie pol olyc yclonal lonal antib ntibodies odies rea react agains gainst Sp Spiropla iroplasma ma

proteins (65), the study must be repeated with monoclonal



The initial studies that appeared suggested that there

were ere defined species bar barriers riers to scrapie infection (66). Ho How- wever

ver, serial passage of infection appears to breakdo breakdown wn those

bar barriers riers (67), again suggesting that the agent is abl able to change

by adaptation or mutation and thus infect a ne new host. Com- Competition

petition has been noted when hen there is exper experimental imental infection

with 2 species of the TSE agent (68). Similarl Similarly, feeding of BSE

of offal fal to other cattle represents essentiall essentially serial passage, which hich

has expe experimentall rimentally led to the increased vir virulence ulence of the agent

(21) and thus spread into the human population as a ne new

variant ariant for form of CJD (69). Both with exper experimental imental Spiroplasma

infection and with scrapie, there is some variable ariable susceptibility

to infection (18, 20). Genetic factors, especiall especially the makeup of

the prion gene, appear to be impor important tant (70), although other

factors such as HLA must be considered (71). In both scrapie

and Spiroplasma infection, neonates are more susceptib susceptible le with

shor shortened tened incubation and a more rapid clinical disease (72).

Dose response is impor important tant in ex experimental perimental Spiroplasma infec- infection,

tion, but this is also related to par particular ticular species of Spiroplasma

(18). ). SMCA induces disease after high-dose inoculum,

whereas hereas GT48 is much more vir virulent, ulent, inducing disease with

much lo lower er titer of inoculum.

In re regard gard to the occur occurrence rence of familial for forms ms of CJD

infection, this could relate to the vertical spread of infection

kno known wn to occur in animals (73). It is note notewor orth thy that another

mechanism ma may in invol volve ve transo transovarian arian passage kno known wn to exi exist st

in Spiroplasma infection in Dr Drosophila osophila (74). In an earl early

study of scrapie in sheep in Texas, exas, the ovaries aries of some of

the animals were ere infectious, which hich is consistent with our hy- ypothesis

pothesis (75). Tateishi ateishi et al ha have ve sho shown wn that in familial for forms ms

of disease, there is a transmissible agent in involv volved ed (76), and

the genetics of the indi individual vidual ma may indicate a cer certain tain suscepti- susceptibility

bility (77, 78).


We already ha have ve suggested that the prion on the surface

of the cell ma may ser serve ve as a receptor for the bacterium. The

question is whe whether ther prion is induced by Spiroplasma and is

responsib responsible for or the disease. ease. We prefer refer the he explanation planation that hat

the prion rion is a reaction eaction product roduct like kely in indu duce ced by argi arginin ine

metalloproteases often associated with patho pathogenic genic bacterial

infection (79), especiall especially because abnor abnormal mal prion isofor isoform is

misfolded at arg arginine inine sites (80). Another possibility is that

Spiroplasma produces RN RNA that facilitates the con conversion version of

nor normal mal PrP PrPc to the protease-resistant for form, m, a mechanism that

has been sho shown wn to be efficient in vitro (81). We fur further ther propose

that the most significant alteration in the tissues relates to

oxyg oxygen en radical damage as evidenced videnced by the prominent accu- accumulation

mulation of products of oxygen radical damage in scrapie and

CJD tissues (22). Spiroplasma thri thrive ve in the presence of oxygen

radicals (82). Neuronal cytoplasmic vacuolization acuolization and mem- membrane

brane fragmentation is seen in exp experimental erimental hypero yperoxia xia in the

rat with accumulation of reaction product of acid phosphatase

acti activity vity (83). It is note notewor orth thy that scrapie infection is agg aggra- ravated

ated by hyperbaric yperbaric oxygen (84). Much of the damage to

tissues of the ner nervous vous system ma may relate to the voracious

appetite of these microbes for lipids and sterols (44).



The most reliable marker for TSE infection is iden- identification

tification of deposits of protease-resistant prion isofor isoforms ms in

the tissues either by immune histochemistr histochemistry or Wester estern bl blot ot

(85). Ho However ver, both protease-sensiti sensitive ve and protease-resistant

for forms of prion on have been een disc discovere ered in TSE, pro probab ably

accounting for absence of prion markers in establ established ished cases of

TSE (86). We ha have ve disco discovered vered a method whereb hereby both for forms ms

of prion can be detected by Wester estern blot (87). In addition,

another protease-resistant fibrillar protein is consistentl consistently found

in TSE tissues and has been refer referred red to as scrapie-associated

fibrils based on the original site of detection (88–90). It is

note notewor orth thy that Spiroplasma contain within them identical

fibril proteins (Fi Fig. g. 5) that are par part of the motility mechanism

of the bacterium (91–93). Spiroplasma fibrils are unique to this

bacterium. Because prion is absent from peripheral blood blood, the

prion is not a likel likely target for de develo veloping ping a peripheral bl blood ood

test for TSE. On the other hand hand, we ha have ve sho shown wn immune

reacti reactivity vity of scrapie antisera with Spiroplasma proteins (65),

and reac eaction tion of TSE SE ser sera with recombin ecombinant ant Spirop piroplasma asma

proteins (data not sho shown) wn) suggest applicability for de devel velop- opment

ment of a peripheral blood test for TSE based on the presence

of Spiroplasma infection.


Spiroplasma infection ser serves ves as a model for TSE in- infection

fection and the fulfillment of Koch’ och’s postulates would ould indicate

that Spiroplasma is the causal agent. Spiroplasma share with

the scrapie agent high infecti infectivity vity, resistance to en environmental vironmental

factors, and lo low immuno immunogenicity genicity. Fur Further ther research based on

immune detection of Spiroplasma-specific proteins could lead

836 q 2005 American Association of Neur Neuropatholo opathologists, gists, Inc.

Bastian J Neuropathol Exp Neurol ! Volume 64, Number 10, October 2005

JOBNAME: jnen 64#10 2005 PAGE: 4 OUTPUT: Tue September 20 15:15:12 2005


to de develo velopment pment of a peripheral bl blood ood diagnostic test for de- detection

tection of subclinical infection in either a transient host or

widespread in the population.


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