Vet. Path.

11: 87-96 (1974)

Toxoplasma-like Encephalomyelitis in the Horse

JILL BEECH and D. C. DODD

School of Veterinary Medicine, University of Pennsylvania, New Bolton Center,

Kennett Square, Pa.

Abstract. Eight horses with progressive neurologic signs had encephalomyelitis asso-
ciated with toxoplasma-like protozoan bodies. There were scattered hemorrhagic, malacic
lesions in white and grey matter in the brain and spinal cord. Microscopically there was
malacia, mononuclear cell infiltration, especially perivascularly, gliosis, and various
degrees of necrosis and hemorrhage. Other tissues were normal, except for the lung of one
horse that had focal bronchopneumonia. The cerebrospinal fluid did not contain measur-
able amounts of IgM, TgG, or TgA. Serum from one horse was negative at I :64 by the
hemagglutination-inhibition test for toxoplasma antibodies.

Toxoplasmosis, a widespread infection and disease, has been reported in

man, laboratory animals, a few feral and zoo animals and many domestic
animals [8]. Tn the English literature there are two reports of disease caused
by toxoplasma in horses [4, 14]. Various researchers [5, 6, 16] have found
evidence of toxoplasma antibodies in serum of different species, including
the horse. Toxoplasma is well known in human infants as a cause of ence-
phalitis subsequent to intrauterine infection and in adults either treated
with immunosuppressive drugs or affected with neoplastic disease. This paper
reports a neurologic disease in eight horses in which toxoplasma-like organ-
isms were associated with and confined to lesions in the central nervous
system.

Clinical Signs

All the horses had neurologic signs that varied in type and severity. Some circled and
had difficulty chewing and swallowing. Four had defects mainly of gait; they dragged their
limbs, especially when backed or pushed to the side, were ataxic, and sometimes knuckled
88 BEECH/DoDD

at a fetlock joint. One horse was slightly hypermetric in a foreleg. Three became recumbent
and had difficulty rising. The course of the disease was usually short. Six were reported
to have had neurologic signs for about 14-21 days, and one progressively worsened and
became recumbent within the course of a month.
The signs recorded in these horses are consistent with those of the well-known clinical
condition called 'segmental myelitis'.

Materials and Methods

Five horses that were examined in the clinic at New Bolton Center were later killed
with overdoses of barbiturate, exsanguinated and necropsied immediately. From three
other horses, either the head or neck only was examined after they were killed. Brains
were removed intact from all horses; in one the cervical cord was also removed, in three
the entire spinal cord, and in one the lumbar cord only. In all but three horses, from which
the head only in two and the neck and head only in one were available, the entire carcass
was examined and sections from major organs taken for microscopic examination. In four
cases, cerebrospinal fluid was aspirated from the cisterna magna for cytologic examination,
cell count, an immunodiffusion test, and assessment of toxoplasma antibody titers.
After fixation in neutral buffered 10% formalin for 3-5 days, sections of brain and
cord were taken at random and from areas with gross lesions. Tissues were cut at 6-8 urn
and stained with hematoxylin and eosin. Many sections were also stained by the Gram,
periodic acid-Schiff (PAS) and Goodpasture methods and with luxol-fast-blue-PAS-
hematoxylin.
Some of the cerebrospinal fluid was collected in an EDTA tube for a leukocyte count,
and some was stored at 4 °C without additive for immunodiffusion and hemagglutination-
inhibition tests for toxoplasma antibodies. Part was diluted I: I with 50% ethyl alcohol
to fix the cells. This sample was centrifuged for 5 min at 1500 rpm, most of the supernatant
decanted, and the remainder placed in a cytocentrifuge for 5 min at 1700 rpm. The slides
were immediately placed in 95% alcohol, fixed for approximately I h, and stained with
Sano's trichrome. In one case, tissue from a site in the cord that was grossly abnormal
was emulsified aseptically and injected intraperitoneally into four mice. After 6 days, the
peritoneal cavities of these mice were flushed with saline, the aspirate examined and
injected into a second set of four mice. This was repeated. Aspirates of fluid from the
cavities of the third set of mice were smeared on slides, air dried, and stained with
Wright's stain.

Results

In all horses significant gross changes were limited to the central nervous
system. In one, of which only the lumbar cord and brain were examined,
there were red-brown foci in the cerebral grey matter. In the other horses
there were scattered yellow areas with punctate red patches affecting prima-
 Equine Protozoan Encephalomyelitis 89

rily white matter on the left side from the fourteenth thoracic to the first
lumbar segment. The entire left half of the cord was yellow and red from
part of the fifteenth to the sixteenth thoracic segment. Other horses had
similar lesions in the brain, one had lesions in the cervical cord, and one had
lesions scattered in the cervical and caudal thoracic cord. In two horses,
the lesions were bilateral yellow and red malacic foci in the internal capsule
and basal ganglia. They extended about 1-3 ern and were about 1.0-1.5 em
in diameter. One horse had a l-cm square gelatinous yellow-grey lesion in
the right basal ganglion; one had several slightly yellow round foci about
0.8 em in diameter and 1.5 em long in the anterodorsal cerebral cortex and
one above the hippocampus extending about I em into the occipital lobe.
Another horse had red and yellow malacic foci in the right internal capsule,
left cerebellar peduncle, on the left side of the anterior medulla and in the
grey substance of the right posterior part of the medulla. One horse with
lesions in both the brain and the cervical spinal cord had soft red and yellow
areas in the right dorsal white matter around the third and fourth cervical
segment; at the cervical enlargement the entire cord was red-brown for
about 2 ern, and proximal and distal to this on alternating sides of the cord
were yellow patches in the white matter for 1-2 ern.
Microscopically the changes were similar in all hOIses. There was extensive
perivascular cuffing (fig. I) with mononuclear cells, mainly lymphocytes.
Eosinophils were always present (fig. 2), but their number varied greatly.
Glial cells and phagocytic cells were less numerous, and there usually were
scattered pyknotic cells and nuclear debris. Malacia (fig. 3, 4) was very
severe in many sections. Except in malacic areas, most of the inflammatory
cells were concentrated around vessels. The amount of hemorrhage (fig. 4),
which was diffuse or focal, varied with anatomical sites and among the
horses. There were various numbers of degenerated neurons. Round ovoid
or elongated pink structures with numerous ovoid dark blue bodies 1-2 urn
long and often closely packed (fig. 5) were scattered throughout the affected
sites. They were usually found only after prolonged search of many sections.
These small bodies were also present in large mononuclear cells (fig. 6), the
identification of which was prevented by distortion of the cellular structure.
The large pink structures, 10-20 urn in diameter, lacked definite walls.
Neither the bodies nor large pink structures stained specifically with PAS,
Gram, or luxol-fast-blue-PAS-hematoxylin stains. It was difficult to differen-
tiate free forms from nuclear fragments although some equivocal ones were
seen. In the spinal cord, in addition to this inflammatory response. there
was vacuolization of the white matter and degeneration and swelling of
90 B EECH /D oDD

•
'" •
I

.
"

•
." • I

Fig, I, Perivascular cuff predom inantl y of lymphocytes. H E.

Fig. 2. Eosinophi ls (arr ows) in cells aro und blood vessel. HE.
 Eq uine Protozo a n Encepha lo mye litis 91

0-

g,
,~
. , I

,
.~

,
" ,-.
•f
:;,
.- ~
,.' ,

I
-. ,
.,
4

Fig . 3. M ulti nuc leat ed ce lls in ma lacic a rea . H E.

Fig. 4. C ircumsc ribed hemo rrh age in ma lac ic a rea . H E.
92 B EECH /D o D D

---
6
••
Fig . 5. Numerou s bodies appa rently free in rnalacic area . H E.
Fig . 6. Cluster of bodies with in cytopl asm of unid enti fiable cell. H E.

ax o ns (fig. 7). In most cas es multinucl eat ed giant cells (fig. 3) were pre sent ;
the number vari ed grea tly from an occasio na l o ne to severa l in alm ost any
field of dam age.
Th e cerebros pinal fluid of fou r horses was negati ve for toxop lasm a a nti-
bod ies at I : 64 by th e hem agglutinati on- inhibiti on test ; by immun od iffusion
tests no a ntibo dies were detected in th e two sa mp les exami ned . Six othe r
 Eq uine Protozo an Encep ha lo mye litis 93

Fig . 7. Swollen axo ns in cr oss a nd longitudinal sectio n in mala cic area . H E.

sa mples fr om normal hor ses and hor ses with other neur ologic diseases not
associ ated with protozoan bod ies were also negativ e. Th e cell counts were
only slightly elevated and the cells were mainly mon onuclear , co nsisting of
sma ll lymph ocytes and la rge histio cytic cells that were occas io na lly bi-
nucleat ed .
T he periton eal fluid of mice inocul ated wit h emulsified cor d was negative
for pr ot ozoa and bacteria.

Discussion

Th e b odie s seen in parts of th e brain and co rd were con sidered to be

pr ot ozoan a nd to resemble th e pr oliferati ve form of toxopl asma .
Th e rea ction in the central nerv ou s system was similar to that described
in other spec ies with toxopl asma ence pha litis except th ere was neither peri -
ventricular necrosis nor ca lcification. Th e bod ies lacked the PAS-positi ve
wall of cystic form s of tox opl asma . In vira l encepha litides th e Iymph ocytic-
mon onuclear reac tio n is sim ilar to th at in our horses, but th e a bsence of
inclusion bodie s, which occ ur in many viral diseases, a nd the ever-pre sent
94 BEECH/DoDD

eosinophils are important points of differentiation. Bacterial and fungal

infections are readily excluded by the failure to demonstrate appropriate
organisms and the absence of a neutrophilic response.
Negative hemagglutination-inhibition tests for toxoplasma titers in the
cerebrospinal fluid do not negate infection but neither do positive titers
confirm it. The antibody content of the cerebrospinal fluid of a dog with
chronic toxoplasmosis was one one-thousandth of the serum titer of I : 32000
[9]. There is no apparent explanation for this difference. These equine cases
were acute, and significant antibody titers in cerebrospinal fluid may not
have had time to develop. In only one case was serum submitted for deter-
mination of toxoplasma antibody. In human infants, results of serologic
studies depend on the tests used and on the infants' ages. Diagnosis of
toxoplasma infection is based on isolation, serology, dermal hypersensitivity,
challenge with a known strain, and histology [9]. We used histology, serology
and isolation. Our single attempt at isolation was unsuccessful. Dermal
hypersensitivity is limited to use in man, guinea pig, and monkey, and,
although it can indicate persistent infection or previous infection, it is not
proof of infection. Challenge of the host would necessitate choosing a
suitable strain; the R H strain is known to present too severe a challenge in
hamsters and mice [8], and it is not known what different strains would do
in the horse. Challenge relies on demonstrating immunity of infected animals
to a heterologous strain of toxoplasma, but this has been found to be a false
assumption at least in mice and hamsters, and, moreover, it is limited to
diagnosis of chronic infections. Histology allows presumptive diagnosis of
toxoplasmosis.
The bodies in the nervous tissue more closely resemble toxoplasma than
other known infective protozoa. The M organism, which was isolated from
field mice in Montana [9, II], is similar to toxoplasma except its cyst is
septate and lobulated. Besnoitea species have been described in horses in
Europa. The disease caused by Besnoitea besnoiti is generalized and often
termed globidiosis. The proliferative form and lesions are identical to those
of toxoplasma, but the cyst form is much larger with a thicker wall, and
the two are immunologically distinct. Encephalitozoon causes generalized
disease and neurologic signs in dogs; lesions are usually disseminated micro-
granulomas affecting many organs, especially the kidney. No lesions other
than those in the nervous system were found in these horses (except the
minor focal pneumonia). Moreover, encephalitozoon is not usually visible
with hematoxylin-eosin stain and is Gram-positive. Nosema, a common
protozoan parasite of insects, can also infect laboratory animals and man
 Equine Protozoan Encephalomyelitis 95

[3], especially persons who have lowered immunologic resistance to orga-

nisms. This parasite is 2 X 4 urn, Gram-positive, sometimes acid-fast, and
has a PAS-positive wall and polar spot. Clearly our parasite is different
from this one, and we conclude that although it most closely resembles
toxoplasma, its identity is unknown.

Acknowledgement

The authors thank Dr. J. P. DUBEY, Department of Pathology, School of Veterinary

Medicine, Ohio State University, for consultation and helpful information about these
cases.

References

ALFORD, C. A.; FOFT, J. W.; BLAKENSHIP, W. J; CASSADY, G., and BENTON, J. W.:
Subclinical central nervous system disease of neonates: a prospective study of infants
born with increased levels of IgM. J. Pediat. 75: 1167-1178 (1969).
2 COHEN, S. N.: Toxoplasmosis in patients receiving immunosuppressive therapy.
J. amer. med. Ass. 211: 657-660 (1970).
3 CONNOR, D. H.; STRANO, A. J., and NEAFIE, R. C.: Nosema. A recently recognized
pathogen of man. Lab. Invest. 30: 371 (1974).
4 CUSICK, P. K.; CELLS, D. M.; HAMILTON, D. P., and HARDENBROOK, H. J.: Toxo-
plasmosis in two horses. J. amer. vet. med. Ass. 164: 77-80 (1974).
5 EYEES, D. E.; GIBSON, C. L.; COLEMAN, N.; SMITH, C. S.;JUMPER,J. R.,and JONES, F.E.:
The prevalence of toxoplasmosis in wild and domesticated animals of the Menthis
region. Amer. J. trop. Med. Hyg. 8: 505-510 (1959).
6 FELDMAN, H. A.: Serological study of toxoplasmosis prevalence. Amer. J. Hyg, 64:
32Q-.325 (1956).
7 FELDMAN, H. A.: Toxoplasmosis. New Eng!. J. Med. 279: 1370-1375 (1968).
8 FRENKEL, J. K.: Host strain and treatment variation as factors in the pathogenesis of
toxoplasmosis. Amer. J. trop. Med. Hyg, 2: 390-411 (1953).
9 FRENKEL, J. K.: Pathogenesis of toxoplasmosis and of infections with organisms
resembling toxoplasma. Ann. N. Y. Acad. Sci. 64: 215-231 (1956).
10 FRENKEL, J. K.: Active immunity to intracellular infection. J. Immuno!. 98: 1309-1319
(1967).
11 FRENKEL, J. K.: Toxoplasmosis: mechanisms of infection, laboratory diagnosis, and
management. Curro Top. Path. 54: 29-75 (1972).
12 GHATAK, N. R.; POON, T. P., and ZIMMERMAN, H. M.: Fine structure of toxoplasma in
the human brain. Arch. Path. 89: 337-348 (1970).
13 KNIGHT, V.: in Textbook of medicine, 13th ed., pp. 736-739 (Saunders, Philadelphia
1971).
14 McDONALD, D. R. and CLEARY, D. J.: Toxoplasma in the equine. SWest. Vet. 23:
212-214 (1970).
96 BEECH/DoDD

15 REMINGTON, J. S.: Toxoplasmosis: recent developments. Annu. Rev. Med. 2/: 201-218
(1970).
16 SATO, N.: Studies on the distribution of dye test antibodies among animals in Hokkaido
and on the complement fixing antigen for toxoplasmosis. Jap. J. vet. Res. 8: 217-218
(1960).
17 SMITH, H. A.; JONES, T. C., and HUNT, R. D.: Veterinary pathology; 4th ed., pp. 164,
168,678, 1111, 1438 (Lea & Febiger, Philadelphia 1972).

Request reprints from: Dr. JILL BEECH, University of Pennsylvania, The School of
Veterinary Medicine, New Bolton Center, R. D. 1, Kennett Square, PA /9348 (USA)

Letters and Comments

Encephalitozoonosis

To the Editor: In the article 'Congenital Encephalitozoonosis in a Squirrel Monkey

(Saimiri sciureus)' by ANVER et al. (Vet. Path. 9: 475-480,1972), the authors cite WOLF
and COWEN (reference II) as having reported congenital encephalitozoonosis in a 4-week-
old female human infant. It has been called to our attention by Dr. J. R. M. INNES that
Drs. WOLF and COWEN later determined that the disease was toxoplasmosis, and this
correction is indicated by INNES and SAUNDERS in Comparative Neuropathology (Academic
Press, New York 1962). Despite this reference, we and others have continued to perpetuate
this erroneous report. We hope that by drawing attention to this error there will be a
reduction in the frequency of this citation in the literature. We also wish to cite a recent
reference to a case of nosematosis in a child: MARGILETH, A. M.; STRANO, A. J.; and
CHANDRA, R.: Disseminated nosematosis in an immunologically compromised infant.
Arch. Path. 95: 145-150 (1973).

R. D. HUNT, M. R. ANVER and N. W. KING

Harvard Medical School
New England Primate Research Center
Southboro, Mass. (USA)