Vet. Pat hoI.

15: 186-1 9 5 (1 978)

A Sarcocystis-like Protozoon in a Sheep with Lymphadenopathy

and Myocarditis

T. L A NDs v ERK , H. G A MLE M a nd R . S VE NK ER U D

Departm ent of Pathol ogy, Vet er inar y Co llege of Norway , Oslo

Abstract. A genera lized e nlarge me nt of the lymph nodes was found in a n e maciat ed
adult e we . Addi tion al a utopsy find ings includ ed tiny grey-white necrot ic foci in th e heart
muscle , aspira tion pneum oni a and diffuse pleuritis. Light microscop y showed a ge ne ralized
lymph ad en op ath y with pc rilyrnph adcn itis, deplet ion of lymph ocytes and histiocytosis of the
lymph nod e . In histiocytes a nd vascular e ndo the lial cells of lymph nod es, se ptal capillary
endot helium of lungs a nd capillary endo the lium of myocardium , ea rly stages of a prot ozoan
para site wer e found. In th e myoca rd ium, ther e we re man y foci of necros is, so me of which
cont ained yo ung cysts in the per iphe ry . Th ese cysts were morph ologically similar to those
of Sarcocystis. Electro n micr oscop y of the ea rly prot ozoan stages yielded evide nce of
schizogo ny a nd form at ion of merozoites.

Sarcocystis' role as a pathogen is being reevalu ated [4] . In experiment al

in fection s the pathogenicity of S . crurl (syn . S . bovicanis) in cattle and S . ovicanis
in s hee p ha s been es ta b lishe d [6 , 8, 11 , 14 , 15] . Anemi a , em aci ation a nd
lymphadenopathy occurred with widespre ad sc hiz og o n y in vascul ar endothelium
[6 , 8 , 11 , 15J . Recent reports of spontan eous acut e sa rco cys tos is in cattl e d es cribe
sim ila r findings [7 , 12]. T he older descript ion of D almen y di se a se is a p p a re ntly
syno ny m o us with sa rco cys tosis [3]. In shee p spo rozoa n- ind uce d enceph alom yelitis
probabl y identical to sa rco cys tos is has be en reported [10] . In this species spo n ta -
neou s ge ne ra lize d infection including lymph adenopath y , howev er , ha s not been
describ ed . This paper reports a spo n ta ne o us ca se of a probable sarcocystis
infection in shee p with lymph adenopath y a nd m yocarditis .

Case History
A 3-year-old G o tla nd ewe had anorexi a and d ebili tation for 2 months . Durin g
th e last 2 we ek s there was a ge ne ra lize d lymph node enl a rgem ent. H a emoglobin
and h aematocrit valu es were norm al , 10 gil 00 ml a n d 31 % re spectivel y , wh ere as
e rythrocyt e counts were slightly decreased - 8 .4 4 x i Oli/mm :l [1 8] . Electrophoresis
of se r u m protein sho we d low tot al prot ein - 4 gil 00 ml se r u m [2] a n d low
ga m ma-g lo b u lin con stitutin g 14 .9 % o f tot al protein [5]. There were 9 800/mm:l
leucocytes; lymphocytes were 59 % and neutrophiles 3 8 % . A lymph node biopsy
18 6

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 Protozoan Lymphadenopathy in Sheep 18 7

fro m the right prescap u lar lym ph nod e was take n . The shee p eve ntua lly had signs
of as piratio n pneu mon ia , die d a nd was a uto psied before a de finit e dia gn osis was
made .

Materials and Methods

Biopsy tissu e fro m the presca p ular lymph nod e a nd a uto psy sec tio ns from me se nt eri c
a nd de e p ingui nal lym p h nod e s , he a rt , live r an d kid ney were processed ro utine ly . T hey
were fixed in Carso n's modified Mi llon igs phosp hate huffered forma lin [11 and stained
wit h hae m a toxylin and eosi n ( HE) a nd va n Gieson . Se lected sectio ns were sta ined with
periodi c acid-Sc hiff ( PAS), Wil d er , G ra m, G ie msa a nd me th yle ne blue . T he o nly ava ilab le
pa ra ffin-em be dd ed spec ime n fro m th e pr e scap ula r ly m ph nod e wa s re hydra te d , fixe d in
osm iu m , deh yd rate d in acetone and e mbedded in ara ldite. T hi n sect io ns ( 1 microm et e r)
were stai ned wit h to lu idine blue for ligh t microscope eval uation . U ltra thin sections for
e lectro n m icr o scop y we re sta ine d with u ran yl aceta te a nd le ad citrate .

Re sults
T he shee p was e macia te d . The lymph nodes were abo ut twice as la rge as
norm al a nd had a moi st , grey , hom ogen o us cut surface. T he right a pica l a nd
ca rdiac lobes of t he lun g had ga ngre no us pne u mon ia . A diffuse se ro fibrino us
pleurit is was see n . In t he poste rio r part of the right dia p hrag matic lobe th e re was
a thin-wa lled abscess, dia met er a bo ut 5 ce nt ime ters , th a t co ntai ned thi ck ye llow
pu s . In th e per icar dial sa c sma ll a mo unts of red serous flu id had accumu la ted . In
th e myoca rdium the re we re man y grey-w hite spots a bo ut 1- 2 mill imet e rs . Th e
live r was pale , t he bile du cts were diste nded and thick-wa lled a nd had man y
Dicrocoelium dendriticum . In th e ca uda te lo be the re were a fe w sma ll abscesses
with a co nte nt simi lar to t hat in th e lung . T he splee n was slightly e nla rged.
Wit h ligh t microsco py t he cha nges in the vario us lymph nod es were basica lly
the sa me. Fa tty tissu e surro und ing the nodes was heavily infilt rat ed with medi um-
sized lym ph ocytes a nd so me macro phages (fig. 1) . T here also was va ried prolife r-
atio n of yo ung gra nulatio n tissu e . Pro tozo a n par asites were ar oun d affe re nt
lymph a tics a nd in t heir endo t helia l cells (fig . 2). Simila r stru ct ures we re in
pericap sul ar tissue (fig. 1) . The co rtica l zo ne was na rrow , a nd fo llicles with
ge rm inal ce ntres were a bse nt. Mature lymphocytes we re depl et ed b ut t he re were
so me med ium to la rge lymphoid ce lls . Hi stiocytic ce lls also had prol ife rated .
A lt ho ug h th e cortical sin uses infreque nt ly had hist iocytes co nta ining prot ozo a , the
med ullar y sinuses were crowde d (fig. 3) . Pr ot ozo an pa rasites also co uld be fo und
in e ndo the lial ce lls of b loo d vesse ls . So me a rteries had a swo lle n medi a wit h
infilt ra tion s of lym ph oid cells in th e adve ntitia where protozoa also co uld be
fo und (fig . 4) . In the medullary re gion th e re was a conspicuo us lymph oi d depl eti on
of the med ullar y co rds .
T he ea rly stages o f th e pa rasites were basophilic , cyto plas mic gra nules (fig . 3).
With pro gr essing ev olutio n t he re was an incr easing basop hilic gr an ular ity of the
cyto plas m fo llowed by agg rega tio n of gran ules int o struct ures rese mb ling nucl e i.
A mo ng t he con de nsed gra nules, there of te n were la rge vac uo les (fig . 3) . Fina lly,

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18 8 La ndsv e rk , Gamlem a nd Svenkerud

Fig. 1: Section from pre scap ula r lym ph no de with thick cap sule (CA ) . Peril ymph atic
tissue ( P L) in filt ra te d by lym ph o cytes a nd so me m acrop hages . O ut er lymph node co rt ex
(C O) with proliferat ion of histio cytes a nd lymph ocytic depleti o n . Mature schizo nt (a rrow).
HE.

th e re was an elaboration of cresce ntic bodie s with a dark basoph ilic nu cleu s a nd a
wea kly stained eosi no p hilic , o r so metimes a mp ho philic, cyto plasm. T hese bodi es ,
co nsidere d to be p rot ozo a , occur red freq uen tly in ro se tt e-like for ma tio n a nd
so me time s see med to be a tta ched to a cen tr al pale bod y . O ccasion all y the re wer e
lar ge ce ntra lly-place d vac uo les . The prot ozo a were e nclosed in a thin ca ps ule a nd
form ed a cyst-like str ucture . T he y were PA S-n egati ve , stained with G ie msa and
toluidin e blu e but o nly wea kly with Gram a nd meth ylen e blu e . In so me a reas th e
cyst wall was arg yro p hilic . T he ea rly stages were clearly intracytopl asmic . Pyknot ic
cha nges in the ho st cell nucl eu s , how ev er , occurred durin g evolution a nd so me-
times th e mature cyst see me d to be free . At times rupture o f th e cyst wall was
see n a nd zo ites were free in tissu e .
In th e myoc ardium there were multipl e foci of dege ne ra tio n a nd calcificati on o f
fibr es (fig . 5) . In so me a reas th ere was o nly a meshw ork o f sa rco le m ma left. The
necrotic foci had a n incipie nt ce llular infiltration con sistin g mainl y of macr oph ages ,
so me lymph oc ytes a nd polym orphonucle ar leu coc ytes; eo sino philes were not
see n . In th e periphery of th e necrotic foci sma ll cysts so me times were fo und (fig .
5) . T he cyst wall was th ick with rad ial spikes similar to tho se of Sarcocys tis . On e
th in-w alle d cyst co ntain ing o nly sphe rical bod ies was see n in a mu scle fib re . In
o the r a reas in th e myoca rdium th ere were lar ge CYSb recognizable as Sarcocystis.
In the ca pilla ry e ndo thelium , pr o tozo an sta ges re sem bling th ose in the lymph

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 Pro to zo an Lymph ad en op ath y in S heep l R9

Fig. 2: Affe rent lymph vessels of prescap ular lymph node with ea rly schizont stages
(arro ws) in endo thelium and in perivascular sites .

no de s occa sion a lly were see n. In th e int erstitium a nd perivascu lar tissue there
were mode rat e to massive infilt ra tio ns th at con sist e d main ly of medium lympho-
cytes .
T he re was hea vy lu ngworm infest ati on o utside ne cro tic a re as in t he lu ngs .
Prot o zo an bo dies were fo und in a cap illar y e ndo t he lial ce ll. In the liver , infla m-
mation of bi le du cts a nd cen troacinar ce ll infiltration ap pa re ntly wa s ca used part ly
by fluk e in fe st ati on . No pro to zo an parasites co uld be fo und in th is o rga n . T he
kidn e y had mo derat e foca l in terstiti a l lymphoc ytic inf ilt ra tio ns , b ut no para sit e s
we re se e n .
T he cresce ntic bo d ies se e n with ligh t microscopy wer e 5- 6 x 1.5- 2 micro me te rs
when se e n with e lec tro n m icr oscopy . T hey ha d a n o ute r thr ee- layered coat o r
pe llicle with o rga ne lles suc h as m icron e mes , rh optri es and co no id a nte rior to the
nucle us ; th e post e rior part had e ndo plas m ic ret iculu m (fig . 6 , 7) . Pol e rin g a nd
sub pe llicula r micro tu bules were indi st inct . T he re wa s he a vy co nd en sati on of
chro ma ti n a lo ng th e nu cle a r me m b ran e . O cc asion a lly micron em es we re fou nd in
bot h ce ll po les . The Go lgi a ppa ra tus a nd especia lly mit och ondri a were not clea r ,
possib ly becau se of the tissue processing . T he num be r of t he m icr o nem e s see med
to in cr e a se with th e age o f the pr ot o zo a , th e yo unger stages cont a ined o nly 30-
4 0 , the ma tu re up to more tha n 120 co unt ed in o bliq ue se ctio ns. T he ab se nce o f
rh o ptrie s in t he yo ung e r stag es wa s co ns picuo us , whereas the bod ie s con tai ni ng

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190 Landsverk, Gamlern and Svenkerud

Fig . 3: Medulla ry a re a o f prescapul ar lymph nod e with ea rly schizo nt sta ges (ES) a nd
o ne mature schizo nt (MS) . Vacu ole s ( V) in yo ung sc hizo nt s . H E .

Fig . 4: Arte ry in the

medullar y a rea of pr escap -
ular lymph nod e . Vacu ola-
tion of medi a a nd de nse
lymphocyt ic infilt ra tio n of
a dve nti tia . Ea rly schizo nts
(a rrows) . H E .

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 Pro to zo a n Ly m p ha de no pat hy in Sheep 19 1

... , .:
~ \

..
.. ' -
'

. ., , . . ..,
..
,.,
"., "

.
"
' • • - t t
\
"
.. ...
.. ....
"

,
'- '
,.
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0'
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Ii ..... . .il
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Fig . 5: Foca l nec ro sis with dystro p hic ca lcifica tio n of myoc a rd iu m , Yo u ng cyst of
Sarcocystis in per ip her y o f nec ro sis (a rro w , inset ) , H E ,

most micr on e mes also had 5- 10 rho pt ries . T he pro tozo a we re e nclo se d in a t hr ee -
laye red cyst wall.
T he yo unges t stage was a n ea rly sc hizogo nic phase wit h nucle i di vided int o
severa l units . T he dividing nucl ei had a n ind e nted nucl ear mem bran e th at ofte n
ga ve t he n ucle i unusua l sha pes . O smiop hilic e nlo ngated bodi es , pr oba bly pr ecur-
so rs of nucl eoli, co nnec ted tw o nucl ear lo be s durin g d ivisio n (fig . 7) . So met imes
clear , thin -w a lled vesi cules a nd can ali cul i we re a mo ng cyto mer-like str uctures and
at th e borde r of t he schizo nt.
T he ne xt de vel o pm ent al stages included elaborat io n of sphe rica l ce lls poor in
o rga ne lles, ex ce pt fo r a few micro ne mes . T he nucl eu s was la rge a nd co nta ine d a
prom inen t nucl eolus a t the bord e r of t he n ucle a r me mbr a ne .

D iscussion
T he fo rm ati on o f me rozo ites in schizo nts wit h th e descr ibed morph ology proves
the spo rozoa n nat ur e of the pa ra site . Of the cocc id ia o nly Toxoplasm a a nd
Sarcocystis have bee n describe d in con nectio n wit h ge ne ra lize d infectio ns in
shee p . O ur o pinio n th at t he repo rt ed pro tozoon is Sarcocystis is based o n the
occ urre nce o f asex ua l sc hizog o n ic stages in va rio us o rga ns o f th e bo dy is we ll
do cume nt ed in Sarcocystis [6 , 8 , 11 , 14 , 15], but not descr ibed o utside ca t

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19 2 Landsverk , Gam lem a nd Svenkerud

...... ~,. ~ .
J "'~ .'\-. f'
"J.' •
( .
""'.r, •
,.
~f.-

Fig. 6: Merozo ites in prescap ular lymph node . C = co no id, ER = endoplas mic re ticu-
lum , MN = micro nc me s , N = n ucleus, PE = pe llicle .

intest ines in Toxo plasma [1 9 , 2 1]. Yo un g deve lop me nta l stages of Sarcocystis
have a pa rtic ula r tro pism for e ndothelial ce lls [6 , 11, 15]. The mor pho logy of the
ma ture me rozoi tes we describe is sim ila r to th at of Sarcocys tis [13 , 20] .
T he focal necrotizing myoca rd itis seen in thi s case rese mb les th at not ed in
experi men ta l infecti on in ca lves [11] . T he lympho id hyp erpl asia se e n in ca lves
[11 , 12] co ntrasts with o ur o bserva tio ns but t he lymph ocyt ic deple tio n of lymph
node s a nd hypogamm aglobul inem ia may indicate impai red im muno log ica l re-
spo nse in o ur shee p.
T here is a conspic uo us d iffe rence of degree of infect io n of o rga ns . The exten sive
pa rasitization of the lym ph nod es an d lymph vesse ls wit h mod e rate infection of

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 Pro tozoan Lymp hadenopa thy in Shee p 193

Fig. 7: Matu re sc hizo nt ( MS) a nd ea rly sch izo nt (ES) in pr e scapu la r lym p h nod e . B =
e lo ngated os miop hi lic bod ies co n nect ing n uclear lo bes . H I = h ist io cyte , M E = mer ozo ite s ,
N I-I = nu cleu s of histiocyte , N = n ucle i o f me ro zoit es , N L = n uclea r lo be s of th e schizo nt,
SM = schizo n t membran e .

lun gs , live r a nd kid ne ys indicate th a t sprea d ing may occ ur t hrough lym ph atic
cha nne ls , altho ugh schizog o ny in vasc ula r e ndo t helium mak es a haematogen ou s
spread prob able .
Po lyart e ri tis nodosa has been fo und regular ly in ass ocia tio n with nosem at osis,
a no the r protozoa n dise ase , in blue foxe s [16 , 17]. T he coex ist en ce o f Sarcocystis
a nd peri arterit is nod osa has pre viou sly bee n not ed in calve s [9]. T he a rte ria l
lesion we describ e a nd th e par asit izati on of vas cula r e ndot he lium rep ort ed by us
a nd ot hers [3 , 6, 7, 11, 12 , 15 ] suggest an ae tio log ica l ro le of cert ain protozoa in
vascula r cha nges in a nima ls .

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 19 4 Lan dsvcr k , Ga m lern a nd Sve nk e rud

Acknowledgements
We th an k D r. H. Mehlh orn , Insti tut fur Zoo Iog i , U n ive rs itiit Du ssel dorf, for his opinio n
o n so me of t he e lec tro n m icro scopi c pic t ures ; t he D e partm ent of Int e rn a l M edicin e I ,
Vet erin ary College of Norway , for clin ica l findin gs , a nd Dr. N . Ek , Ve terinary College o f
No rway , for bl o od pro tei n dat a.

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Reque st reprint s from T ho r Landsverk , Dep artm ent o f Pathology, Veterinary Co lleg e of
No rway, P.O . Box 11 146 , Os lo-Dep ., Os lo 1, No rway.

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