94 Brief communications
contaminating the serum, possibly due to collection tech-
nique. The sera in group B were collected by draining the
contents of the umbilical cord into glass tubes. It is quite
possible that HI substances in fetal fluids could contaminate
the serum samples, resulting in apparent specific HI activity
for EMCV and accounting for the 3 positive (124) titers in
group B serum samples.
Several studies have evaluated fetal serum for the presence
of Ig.4,11,13 The reports describe small amounts of Ig present
that may or may not be functional at birth. The protein A
precipitation of Ig agrees with previous studies in indicating
that little if any detectable Ig is present in naive immuno-
competent fetuses.
Although the in vitro assays are specific tests for the pres-
ence of antibody-antigen interactions, the lack of evidence
supporting serum EMCV antibody and the apparent sero-
conversion of dead fetuses indicates that these specific tests
can result in false positives. These findings emphasize the
importance of critically evaluating fetal serology, especially
when body fluids other than fresh serum are used as the test
sample. Caution should be exercised when using TF as the
sole means of diagnosing EMCV-related porcine fetal death.
Acknowledgement. We thank Deb Adolphson for tech-
nical assistance. No endorsements are herein implied. Brand
names are necessary to report factually on available data;
however, the USDA neither guarantees nor warrants the
standards of the products, and the use of the names by the
USDA implies no approval of the products to the exclusion
of others that may also be suitable.
Sources and manufacturers
a. Kimble, Toledo, OH.
b. Dr. P. S. Paul, College of Veterinary Medicine, Iowa State Uni-
versity, Ames, IA.
References
1. Christianson WT, Kim HS, Joo HS, Barnes DM: 1990, Re-
productive and neonatal losses associated with possible enceph-
alomyocarditis virus infection in pigs. Vet Rec 126:54-57.
J Vet Diagn Invest 594-97 (1993)
Poliomyelomalacia and ganglioneuritis in a horse with paralytic rabies
Donal O’Toole, Kenneth Mills, John Ellis,
Val Welch, Mel Fillerup
Rabies is one of the classical viral encephalitides of humans
and domestic animals, caused by a rhabdovirus of the genus
Lyssavirus. Morphological lesions of rabies in the neuraxis
of infected animals vary in severity and location and are
From the Wyoming State Veterinary Laboratory, 1174 Snowy
Range Road, Laramie, WY 82070 (O’Toole, Mills, Ellis, Welch),
and Cody Animal Health and Supply, 2023 Sheridan Avenue, Cody,
WY 82414 (Fillerup).
Received for publication January 25, 1992.
2. Dea SA, Bilodeau R, Martineau GP: 1991, Isolation of enceph-
alomyocarditis virus among stillborn and post-weaning pigs in
Quebec. Arch Virol 117: 121-128.
3. Dea S, Bilodeau R, Sauvageau R, Martineau GP: 1991, Out-
breaks in Quebec pig farms of respiratory and reproductive
problems associated with encephalomyocarditis virus. J Vet
Diagn Invest 3:275-282.
4. Franek F, Riha I, Sterzl J: 1961, Characteristics of gamma
globulin, lacking antibody properties, in new-born pigs. Nature
189:1020-1022.
5. Joo HS, Kim HS, Leman AD: 1988, Detection of antibody to
encephalomyocarditis virus in mummified or stillborn pigs. Arch
Virol 100: 131-134.
6. Kim HS, Joo HS, Bergeland ME: 1989, Serologic, virologic,
and histopathologic observations of encephalomyocarditis virus
infection in mummified and stillborn pigs. J Vet Diagn Invest
1:101-104.
7. Kim HS, Joo HS, Christianson WT, Morrison RB: 1991, Eval-
uation of serologic methods for the detection of antibodies to
encephalomyocarditis virus in swine fetal thoracic fluids. J Vet
Diagn Invest 3:283-286.
8. Links IJ, Whittington RJ, Kennedy DJ, et al.: 1986, An as-
sociation between encephalomyocarditis virus infection and re-
productive failure in pigs. Aust Vet J 63: 150-152.
9. Mengeling WL: 1972, Porcine parvovirus: properties and prev-
alence of a strain isolated in the United States. Am J Vet Res
33:2239-2247.
10. Mengeling WL, Lager KM: 1991, Serologic diagnosis of porcine
prenatal and neonatal infections: comparison of thoracic fluids
and sera for detecting antibodies and immunoglobulin. Proc
Livest Conserv Inst, pp. 129-132.
11. Prokesova L, Kostka J, Rejnek J, Travnicek J: 1970, Further
evidence of active synthesis of immunoglobulins in precolostral
germfree piglets. Folia Microbiol 15: 337-340.
12. Sanford SE, Josephson GISA, Rehmtulla AJ, Carman PS: 1989,
Antibodies to encephalomyocarditis virus in aborted and still-
born pigs. Can Vet J 30:757.
13. Sterzl J, Kostka J, Riha I, Mandel L: 1960, Attempts to de-
termine the formation and character of gamma globulin and of
natural and immune antibodies in young pigs reared without
colostrum. Folia Microbiol 5:2944.
generally microscopic in both animals14 and humans1,11 In
some cases of rabies, mild cerebral edema or meningeal con-
gestion can be appreciated grossly.1,11 Striking gross lesions
have rarely been documented.3,14 This is a report of such a
lesion in the spinal cord of a horse.
A 6-year-old castrated quarterhorse developed sudden on-
set posterior paresis that progressed over the following 7 days
to quadriparesis and recumbency. There was no history or
evidence of a recent bite wound. Cerebrospinal fluid, col-
lected with xyalzine/ketamine hydrochloride restraint, was
 Brief communications
Figure 1. Bilateral hemorrhage and malacia in gray matter of formalin-fixed spinal cord of a horse.
normal when examined on day 3. In addition to urinary
bladder paralysis, the horse developed hypalgesia, hypotonia,
and hyporeflexia of the tail, anus, and perineum. Treatment
comprised dihydrostreptomycin/penicillin G procaine, B vi-
tamins, trimethoprin/sulfadiazine, dimethyl sulfoxide in lac-
tated Ringer’s solution, flunixin meglumine, oral electrolyte
solution, sodium selenite/vitamin E, and dexamethasone. No
evidence of behavioral change or altered consciousness was
manifested until day 7, when the horse stopped eating, de-
veloped pitting edema of hind legs and perineum, became
depressed, and was mildly aggressive. The clinical diagnosis
was equine herpesvirus (EHV-1) myeloencephalitis, with a
differential diagnosis including equine protozoa1 myelitis, in-
toxication, and rabies. The horse was euthanized with an
overdose of sodium pentobarbital on day 7 and immediately
examined.
Complete spinal cord, half of the brain, and samples of
kidney, urinary bladder, and lung were submitted in formalin
to the Wyoming State Veterinary Laboratory for microscopic
examination. The other half of the brain was submitted un-
fixed. Impression smears were made of unfixed hippocam-
pus, were incubated with fluorescein-conjugated antirabies
antibodies,a and were examined by fluorescence microscopy.
Impression smears were also made of caudal medulla ob-
longata and were incubated with fluorescein-conjugated an-
tibodies to EHV- 1.b Virus isolation was not attempted. Mul-
tiple levels of formalin-fixed spinal cord (including dorsal
root ganglia [DRG] and spinal nerve roots), brain, and sam-
ples of kidney, bladder, and lung were processed for hema-
toxylin and eosin (HE)-stained sections. Formalin-fixed par-
affin-embedded sections of brain and spinal cord were
examined using an avidin-biotin complex (ABC) immuno-
cytochemical technique6 and lapine anti-rabies virus anti-
bodies.c Control tissue for the ABC technique comprised
sections of confirmed positive rabies virus-infected brain and
test sections of the horse’s neuraxis where the primary an-
tibody was either omitted or replaced by normal lapine se-
rum. Formalin-fixed pieces of cerebellum, medulla oblonga-
ta, and DRG (one site) were diced into 1 -mm3 pieces, postfixed
in glutaraldehyde and osmium tetroxide, and processed for
semithin (1 µm) and ultrathin (70-90 nm) sections. Spinal
cord gray matter was not examined ultrastructurally because
malacia was so extensive. Ultrathin sections were stained
and examined using a Philips 410 electron microscope.
Gross lesions were restricted to the spinal cord and urinary
bladder. There was severe diffuse bilateral poliomyeloma-
lacia with or without hemorrhage throughout the spinal cord
(Fig. 1). There were no gross abnormalities in the brain.
Multifocal hemorrhagic and ulcerative cystitis was also pres-
ent.
Fluorescent antibody (FA) examination of smears of hip-
pocampus and medulla oblongata for rabies virus and EHV-
96 Brief communications
Figure 2. Section of dorsal root ganglion of a horse, with non-
suppurative ganglioneuritis, neuronal degeneration, and satellitosis
(Van Gehuchten and Nelis lesion). HE stain. Bar = 50 µm.
1, respectively, were negative. Histopathologic examination
revealed widespread malacia of spinal cord gray matter. In-
filtrates of gitter cells and mild perivascular cuffs of histiocytic
and lymphocytic cells occurred at the margins of malacic
areas. Many remaining spinal cord neurons had hyalinized
cytoplasm and crenated, eccentric nuclei. White matter
changes in spinal cord were mild, comprising occasional his-
tiocytic-lymphocytic perivascular cuffs. All DRG examined
had severe diffuse lymphocytic infiltration (Fig. 2). Some
individual neurons had large intracytoplasmic vacuoles.
Neuronophagia and satellitosis were widespread in DRG.
Mild multifocal nonsuppurative encephalitis with gliosis was
present in medulla oblongata. Changes in more rostra1 areas
of brain were minimal. Negri bodies were absent in brain
and spinal cord. Rabies viral antigen-positive intracyto-
plasmic inclusions were detected in ABC preparations in
descending frequency in medulla oblongata, spinal cord gray
matter, cerebellum, thalamus, and DRG. Inclusions were
small (< 1-3 µm), sparse, and usually located at the periphery
of perikarya (Fig. 3) and in neurites. Electron microscopic
examination of brain and DRG did not reveal virus-like
particles.
A distinctive feature of this case was the occurrence of
grossly evident malacia and hemorrhage in gray matter of
the spinal cord. Gross lesions have been reported as minimal
or absent in the neuraxis of rabid animals. 14 Grossly evident
hemorrhagic changes in gray matter of equine spinal cord,
similar to those seen in this case, have however been doc-
umented,14 and severe necrosis of equine spinal cord gray
matter is sometimes evident histologically.7 Unlike most
Figure 3. Immunocytochemical preparation of spinal cord gray
matter of a horse. Small viral antigen-positive inclusions (arrow-
heads) are predominantly at the cytoplasmic margins of the peri-
karyon in this alpha motorneuron. ABC technique, 1:4,000 dilution
of primary serum. Bar = 10 µm.
rhabdoviruses, rabies virus is not itself directly cytolytic,12
and neuronal degeneration in infected brains is generally
minima1.2,9 Rabies virus infection can however result in ex-
tensive cell death in vitro if infected cells are exposed to
specific antibody and complement during periods of peak
viral maturation at the plasmalemma.8,18 There is a particular
association between ascending myelitis and bites from rabid
hematophagus bats.7 Unfortunately, no unfixed tissue was
retained from this case for virus isolation and typing by
monoclonal antibody panels13 to establish whether an un-
usual strain of rabies virus was involved.
Ganglioneuritis (Van Gehuchten and Nelis lesion) is help-
ful in making a tentative diagnosis of rabies particularly when,
as in this case, FA results for rabies are negative and Negri
bodies are absent.7 Other authors have emphasized the value
of examining autonomic ganglia, particularly Gasserian and
dorsal root ganglia,5 and have considered the ganglioneuritis
sufficiently characteristic to permit presumptive diagnosis of
rabies when Negri bodies could not be found.7 No virus was
detected ultrastructurally, and the amount of viral antigen
detected on immunocytochemistry was small, so the basis
for the florid inflammatory and degenerative change in DRG
is unclear. Attempted ultrastructural detection of rabies virus
in DRG in positive cases can be unrewarding, even when
ganglionitis is marked. 15 Ganglial ensconcement has been
implicated as a means of protracted viral incubation in ra-
bies,12 and neurons in autonomic ganglia are peculiarly in-
efficient in producing progeny rabies virus. 10,17 Neuronal vac-
 Brief communications
uolation in DRG, as seen in this horse, is a feature of some
cases of rabies7 and spongiform change, beginning as vac-
uolation principally in dendrites, is a significant aspect of the
histopathology of rabies.2
Direct FA examination is regarded as the best available
test for a rapid and specific diagnosis of rabies.16 In this case,
the brain was FA negative, probably because only hippocam-
pus, and not brain stem, was examined. Brain stem and
cerebellum provide the best diagnostic tissues for direct FA
16
testing of livestock. Diagnosis was achieved by immuno-
cytochemistry, a method of growing importance in both re-
search and diagnostic aspects of rabies.2,4,9,19 Mouse inocu-
lation continues to be an important method for confirmation
of a diagnosis of rabies, with a detection rate of 95%. Rabies
immunocytochemical examination of formalin-fixed tissue
can be as sensitive as FA,19 with the advantage that it can
be used when the only tissue available is formalin or glutar-
aldehyde fixed or when autolysis, which compromises both
FA examination16 and attempted virus isolation in cell cul-
ture,16 is present.4
Acknowledgements. We appreciate the confirmation of
rabies in this case by Dr. D. M. Haines and the immuno-
cytochemistry unit at the Western College of Veterinary Med-
icine, Saskatoon, Saskatchewan, Canada, using an ABC tech-
nique. We thank Mary Benson for typing the manuscript.
Sources and manufacturers
a. Becton Dickinson, Cockeysville, MD.
b. National Veterinary Services Laboratory, Ames, IA.
c. Dr. K. M. Charlton, Agriculture Canada, Animal Diseases Re-
search Institute, Nepean, ON, Canada.
References
1. Adams JH, Corsellis JAN, Duchen LW: 1984, Rabies. In:
Greenfield’s neuropathology, eds. Hume Adams J, Corsellis Jan,
Duchen LW, 4th ed., pp. 280-282. John Wiley & Sons, New
York, NY.
2. Charlton KM, Casey GA, Webster WA, Bundza A: 1987, Ex-
perimental rabies in skunks and foxes. Pathogenesis of the spon-
giform lesions. Lab Invest 57:634-645.
3. Dolman CL, Charlton KM: 1987, Massive necrosis of the brain
in rabies. Can J Neurol Sci 14: 162-165.
4. Feiden W, Kaiser E, Gerhard L, Dahme E, Gylstorff B, Wandeler
J Vet Diagn Invest 5:97-99 (1993)
Serologic responses to the mallein test for glanders in solipeds
Joyce M. Hagebock, Linda K. Schlater, Wayne M. Frerichs, David P. Olson
Glanders is a contagious disease of solipeds caused by
Pseudomonas mallei; humans and carnivores are accidental
hosts.1,2,12 The disease is usually chronic in horses, and an
From the US Department of Agriculture, Animal and Plant Health
Inspection Service, Veterinary Services, National Veterinary Ser-
vices Laboratories, Ames, IA 50010.
Received for publication April 3, 1992.
97
A, Ehrensberger F: 1988, Immunohistochemical staining of
rabies virus antigen with monoclonal and polyclonal antibodies
in paraffin tissue sections. J Vet Med B 35:247-255.
5. Frothingham L: 1920, Something about glanders and rabies.
Cornell Vet 10:163-173.
6. Haines DM, Chelack BJ: 1991, Technical considerations for
developing enzyme immunohistochemical staining procedures
on formalin-fixed paraffin-embedded tissues for diagnostic pa-
thology. J Vet Diagn Invest 3: 101-112.
7. Innes JRM, Saunders LZ: 1962, Rabies. In: Comparative neu-
ropathology, eds. Innes JRM, Saunders LZ, pp. 384-394. Aca-
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8. Iwasaki Y, Wictor TJ, Koprowski H: 1973, Early events of
rabies virus replication in tissue cultures. An electron micro-
scopic study. Lab Invest 28:142-148.
9. Jackson AC, Reimer DL, Ludwin SK: 1989, Spontaneous re-
covery from the encephalomyelitis in mice caused by street
rabies virus. Neuropathol Appl Neurobiol 15:459-475.
10. Lycke E, Tsiang H: 1987, Rabies virus infection of cultured rat
sensory neurons. J Virol 61:2733-2741.
11. Perl DP, Good PF: 1991, The pathology of rabies in the central
nervous system. In: The natural history of rabies, ed. Baer GM,
2nd ed., pp. 163-190. CRC Press, Boca Raton, FL.
12. Rupprecht CE, Dietzschold B: 1987, Perspectives on rabies
virus pathogenesis. Lab Invest 57:603-606.
13. Smith JS, Reid-Sanden FL, Roumillat LF, Trimarchi C, Clark
K, Baer GM, Winkler WG: 1986, Demonstration of antigenic
variation among rabies virus isolates using monoclonal anti-
bodies to nucleocapsid proteins. J Clin Microbiol 24:573-580.
14. Sullivan ND: 1985, The nervous system. In: Pathology of do-
mestic animals, ed. Jubb KVF, Kennedy PC, Palmer N, 3rd ed.,
vol. 1, pp. 293-296. Academic Press, Orlando, FL.
15. Sung JH, Hayano M, Mastri A, Okagaki T: 1976, A case of
human rabies and ultrastructure of the Negri body. J Neuro-
pathol Exp Neurol 35:541-559.
16. Trimarchi CV, Debric JG: 1991, The fluorescent antibody in
rabies. In: The natural history of rabies, ed. Baer GM, 2nd ed.,
pp. 220-233. CRC Press, Boca Raton, FL.
17. Tsiang H, Derer M, Taxi J: 1983, An in vivo and in vitro study
of rabies virus infection of the rat superior cervical ganglia. Arch
Virol 76:231-243.
18. Wiktor TJ, Clark HF: 1972, Chronic rabies virus infection of
cell cultures. Infect Immun 6:988-995.
19. Zimmer K, Wiegand D, Manz D, Frost JW, Reinacher M, Frese
K: 1990, Evaluation of five different methods for routine di-
agnosis of rabies. J Vet Med B 37:392-400.
acute form is often seen in donkeys, asses, and mules. The
nasal, pulmonary, and cutaneous forms (farcy) are distin-
guishable by the location of the lesions. However, all 3 forms
may occur simultaneously in 1 animal.2 The prognosis in
infected animals is generally unfavorable. Recovered animals
usually do not develop immunity. These and latently infected
animals are a source of infection to other equine populations
and to humans, in whom the disease is sometimes fatal.