Dubey 1996

veterinary
parasitology
ELSEVIER Veterinary Parasitology 67 (1996) 1-59
Review
A review of Neospora caninum and neosporosis

J.P. Dubey a,*, D.S. Lindsay b
a Parasite Biology and Epidemiology Laboratory, Livestock and Poultry Sciences Institute, Agricultural
Research Service, US Departmentof Agriculture, BARC-East, Beltsville, MD 20705-2350, USA
b Departmentof Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519,
USA
Received 15 February 1996; accepted 2 May 1996
Abstract
Neospora caninum is a recently recognized protozoan parasite of animals, which until 1988
was misidentified as Toxoplasma gondii. Its life cycle is unknown. Transplacental transmission is
the only recognized mode of transmission. It has a wide host range, but its zoonotic potential is
unknown. Neosporosis is a major cause of abortion in cattle in many countries. It is also an
important cause of neuromuscular paralysis in dogs. This paper reviews information on parasite
structure, life cycle, biology, clinical signs, diagnosis, treatment and control.
Keywords: Neospora caninum; Neosporosis;Tachyzoites, Tissue cysts; Abortion; Paralysis; Animals
1. Introduction and history

Neospora caninum is a recently recognized protozoan parasite of livestock and
companion animals. Since the description of the parasite in 1988 (Dubey et al., 1988a),
over 100 papers have been published on this subject. Because of the prevalence and
economic importance of neosporosis in dogs and cattle, we will focus this review on
these animals. Historic events are summarized in Table 1.
2. Structure and biology

2.1. Structure
Tachyzoites and tissue cysts are the only life cycle stages identified to date.
Tachyzoites are ovoid, lunate or globular and measure 3 to 7 1 to 5 / z m , depending on
* Corresponding author. Tel: 301-504-8128;fax: 301-504-9222;e-mail: JDUBEY@GGPLARSUSDA.GOV.
0304-4017/96/$15.00 Published by Elsevier Science B.V.

PI! S0304-4017(96)01035-7
2 J.P. Dubey, D.S. Lindsay/Veterinary Parasitology 67 (1996) 1-59
Table 1
History of Neospora caninum and neosporosis
Contribution Reference
1. Disease first recognized in dogs in Norway, Bjerk~is et al. (1984)
but not named.
2. New genus, Neospora and the type species N. caninum Dubey et al. (1988a)
proposed for the protozoan from dogs in USA.
3. Neospora caninum isolated in cell culture and Dubey et al. (1988b)
mice and Koch's postulates fulfilled.
4. Indirect fluorescent antibody test developed Dubey et al. (1988b)
for serologic diagnosis of neosporosis.
5. Immunohistochemical test developed to identify Lindsay and Dubey (1989c)
Neospora organisms in tissues.
6. Neoporosis identified as cause of abortion in Thilsted and Dubey (1989)
dairy cattle.
7. Transplacental transmission of N. caninum induced Dubey and Lindsay (1989a,b,1990b);
in dogs, cats, sheep, and cattle. Dubey et al. (1992c)
8. Experimental models for neosporosis developed Lindsay and Dubey ( 1989d, 1990c)
in mice and rats.
9. Drags screened for chemotherapy of neosporosis. Lindsay and Dubey ( 1989b, 1990a)
10. The Norwegian dog parasite identified as N. caninum. Bjerk/is and Dubey ( 1991)
I 1. Neosporosis recognized as a major cause of Anderson et al. (1991); Barr et al. (1991a)
bovine abortion in California drylot dairies.
12. Neo~pora isolated from bovine aborted fetuses and Conrad et al. ( 1993a); Barr et al. (1994b)
disease induced in cattle with bovine isolate.
13. Neo6pora caninum shown to be a common Par6 et al. (1994)
asymptomatic infection in dairy cattle.
14. ELISA developed for diagnosis of neosporosis Bj6rkman et al. (1994a); Par~ et al. (1995b);
in dogs and cattle, Dubey et al. (1996a)
15. First recombinant N. caninum proteins produced Lally et al. (1996a)
for diagnosis.
the stage of division. They divide into two zoites by e n d o d y o g e n y (Fig. 1). In infected
animals, tachyzoites are found in m a n y cells including neural cells, macrophages,
fibroblasts, vascular endothelial cells, myocytes, renal tubular epithelial cells and
hepatocytes ( D u b e y et al., 1988a; D u b e y , 1993a; Bjerk&s and Presthus, 1989; Speer and
Dubey, 1989). Infected host cells m a y contain as m a n y as 100 tachyzoites in one plane
of a section.
Tachyzoites penetrate host cells by active invasion and can b e c o m e intracellular
within 5 m i n o f contact with host cells (Hemphill et al., 1996). Tachyzoites are usually
located within the host cell cytoplasm within a parasitophorous vacuole (pv). Tachy-
zoites may be located in more than 1 pv in a host cell. In some electron micrographs
from in vivo infected cells a true pv could not be identified ( D u b e y et al., 1988a; Speer
and Dubey, 1989); whether this observation is an artifact remains to be determined. Few,
m a n y or no intravacuolar tubules m a y be present in the pv (Bjerk&s and Presthus, 1988,
Bjerk/is and Presthus, 1989; Speer and D u b e y , 1989). N e o s p o r a caninum tachyzoites
have a three layered p l a s m a l e m m a , 22 subpellicular microtubules, two apical rings, a
conoid, a polar ring, mitochondria, up to 150 micronemes, eight to 18 rhoptries, some
J.P. Dubey, D.S. Lindsay / Veterinary Parasitology 67 (1996) 1-59 3
Fig. 1. Neospora caninum tachyzoites in human foreskin fibroblast cells in culture. A to D, light microscope,
methanol f'uted, Giemsa stain; Ba~. 10 p,m. Tachyzoites are located in parasitophorous vacuoles (pv). A.
Dividing tachyzoites with one end still attached to a residual body (arrow). B. Pair inside a pv. C. Four
tachyzoites attached to a residual body (arrow). D. Sixteen tachyzoites in a p v . E. Transmission electron
micrograph. Tachyzoite with a conoid (c), several micronemes (mi), a mitochondrium (m0, a nucleus (n), and
seven rhoptries (r), one of which is extending almost to the posterior end of the nucleus. Bar:. 0.50 /.~m. F.
Transmission electron mierograph. Divided tachyzoites. Note only a few rhoptries (r), micronemes (rni) and
tubular network in a pv. Bar. 0.57/~m.
extending posterior to the nucleus (Fig. 1E), a Golgi complex, rough and smooth
endoplasmic reticulum, a nucleus and a nucleolus (Speer and Dubey, 1989; Lindsay et
al., 1993) features which are not dependent on the isolate of N. caninum (Fig. 1E, F).
The number of micronemes is highly variable. Some of the micronemes may be
perpendicular to the inner parasite membrane (Fig. 1E, F). The rhoptries contain solid
electron dense material and are two to four times thicker than the diameter of the
micronemes. The discrepancy in the number of rhoptries reported by different re-

searchers may be due to the difficulty in distinguishing rhoptries from dense granules
(Bjerk[is et al., 1984; Dubey, 1993a). In some groups of tachyzoites, the matrix between
the individual parasites, is electron-dense and contains free micronemes and collections
of tubular structures (Dubey et al., 1995). Micropores have not been seen in tachyzoites
in animals, but were found in tachyzoites grown in cell culture (Speer and Dubey, 1989;
Lindsay et al., 1993).
Tissue cysts are often round to oval in shape, up to 107 ~ m long and have been
observed only in neural tissues (brain, spinal cord, nerves and retina) (Dubey et al.,
1988a; Dubey et al., 1990d) with the single exception of a solitary tissue cyst in the
ocular muscles of a foal (Lindsay et al., 1996c). The tissue cyst wall is smooth and up to
4/~m thick, presumably depending upon how long the infection has existed (Figs. 2 and
3). In most tissue cysts the cyst wall is 1 to 2/xm thick. The cyst wall contains branched
tubule-like structures (Bjerk~s and Dubey, 1991). Septa are absent and there is no
secondary cyst wall (Fig. 4). Bradyzoites are slender (6-8 1-1.8 /zm) and contain the
!
! i!)
Fig. 2. Neospora caninum tachyzoites and tissue cysts in sections of CNS of naturally infected cattle. Bar: 10
/.tm and applies to A to G.. A: immunohistochemical stain, B to G: H&E stain. A. Two tachyzoites
(arrowheads) among necrotic host cells. B. A group of extracellular well preserved tachyzoites (arrowheads).
C, lntracellular tachyzoites (arrowhead) m a neuron. D. Youngest recognizable tissue cyst containing eight
bradyzoites, The cyst wall is very thin (arrow). E. Tissue cyst with 12 bradyzoites and still with a thin cyst
wall (arrow). F. Tissue cyst in a neuron. The cyst wall (arrow) is thick but bradyzoites are not very distinct. G.
Tissue cyst with numerous elongated bradyzoites enclosed in a thick cyst wall (arrow). The host cell nucleus is
at the lower end.
J.P. Dubey, D.S, Lindsay / Veterinary Parasitology 67 (1996) 1-59 5
Fig. 3. Tissue cyst in brain squash of a mouse 12 months after inoculation with N. caninum. NoI~ the thick
cyst wall (opposing arrowheads) enclosing slender bradyzoites (arrow). Unstained; Bar: 10 p,m.
same organelles as are found in tachyzoites except that there are fewer rhoptries and
more periodic acid Schiff (PAS)-positive (amylopectin) granules in the bradyzoites. The
nucleus in bradyzoites is terminal to subterminal in location (Fig. 4). The micronemes in
bradyzoites are often arranged perpendicular to the plasmalemma. The cyst wall stains
variably with PAS and is argyrophilic (Dubey, 1993a). Tubular vesicular structures are
present between bradyzoites (Bjerk~s and Presthus, 1989; Bjerl~ and Dubey, 1991).
Micropores are rare (Bjerk~s and Dubey, 1991; Speer and Dubey, 1989; Jardine, 1996).
Recently, Jardine (1996) described vesiculo-membranous organelles containing short,
flat membranous segments and smaller vesicles in bradyzoites.
2.2. Life cycle
Neospora caninum has a wide host range. Evidence of natural infections has been
found in dogs, cattle, sheep, goats, horses, and deer. Experimental infections have been
induced in mice, rats, dogs, foxes, goats, cats, sheep, coyotes, pigs, gerbils, rabbits,
sheep, and cattle (for citations see appropriate sections).
The life cycle of N. caninum is unknown. A transplacental route is the only
recognized mode of transmission. Transplacental infection has been induced experimen-
tally in dogs (Dubey and Lindsay, 1989b; Cole et al., 1995b), cats (Dubey and Lindsay,
1989a), sheep (Dubey and Lindsay, 1990b; McAllister et al., 1996b), cattle (Dubey et
al., 1992c; Barret al., 1994b), and mice (Cole et al., 1995a; Long and Baszler, 1996).
Fig. 4. Transmission electron micrographs of a tissue cyst in the brain of a naturally infected dog. A.
Relatively thin cyst wall (opposing arrows) enclosing numerous bmdyzoites; Bar: 1.66 /xm. B. Bradyzoite
with a micmpore (rap), few rhoptries (r), and several micronemes (mi), some of them are arranged
perpendicular to the plasmalemma. Note a subterminal nucleus (n). Bar: 0.83 /zm. (Courtesy of Bjerk/is, I.,
Norwegian College of Veterinary Medicine, Oslo, Norway).
Transplacental infection can occur repeatedly in the same animal (Bjerk~ts et al., 1984;
Dubey et al., 1988b; Barr et al., 1993). Experimental N. caninum infection in a cat
before pregnancy has led to congenital infection in a kitten (Dubey and Lindsay, 1989a).
Neospora caninum tachyzoites were found in the placenta and the uterus of the queen
(Dubey and Lindsay, 1989a). In animals inoculated experimentally, N. caninum is
infective by the subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), intravenous
(IV) and oral routes. Bradyzoites in tissue cysts were resistant to HCl-pepsin solution,
indicating that camivorism may be a part of the life cycle of N. caninum (Lindsay and
Dubey, 1990b). Neospora caninum tissue cysts can survive up to 14 days at 4C, but are
noninfective after incubation at - 2 0 C for 1 day (Lindsay et al., 1992). Oral infections
of mice with tachyzoites may have been secondary to oral pharyngeal trauma and do not
necessarily represent gastrointestinal invasion by tachyzoites. In one instance, Neospora
in calf brain survived freezing at - 52C (Bryan et al., 1994). Recently, McGuire et al.
(1996) developed a method to separate and freeze N. caninum tissue cysts from mouse
brains.
A carnivore definitive host is suspected in the life cycle of N. caninum. However, in
experiments in our laboratory, oocysts have not been found in feces of cats, dogs,
coyotes, raccoons, and carnivorous birds including red tailed hawks, turkey vultures,
crows, and barn owls given N. caninum infected tissues (Table 2). These results are not
definitive because there is no reliable way to obtain tissue cysts of N. caninum in an
experimental host. In experiments in Table 2, the presence of tissue cysts in the
inoculum given to most animals was not ascertained. However, we feel confident that
cat is not the definitive host for N. caninum. In addition to data in Table 2, Bjerklts
(1992) mentioned that oocysts were not found in feces of foxes, but no details were
given.
Based on circumstantial evidence, Hammondia pardelis (with an lsospora felis-like
oocys0 oocysts were considered as a stage of N. caninum by Abbitt et al. (1993).
However, these findings were questioned by Dubey (1993b), and McAllister (1993).
Hammondia pardelis oocysts were seen in feces of cats fed muscles from cows that had
aborted due to neosporosis. Because this work was done in 1984, before the discovery of
N. caninum, the identity of oocysts in feces of cats as a stage of N. caninum could not
be established.
2.3. In vitro cultivation
Neospora caninum was initially cultured in vitro in bovine monocytes and bovine
cardio-pulmonary arterial endothelial cells, with more organisms being produced in the
former (Lindsay and Dubey, 1989a). Since that time N. caninum has been grown in
Madin-Darby bovine kidney, human foreskin fibroblast, Vero cells, several other well
established cell lines and fetal mouse brain (Fig. 1). Some isolates of N. caninum
multiply faster than others and interferon gamma can inhibit intracellular multiplication
of tachyzoites (Innes et al., 1995). Only tachyzoites have been identified in cultivated
cells, and organisms from cell cultures are infective for animals. Continuous serial
passage of tachyzoites for 8 years in cell cultures has been achieved with no loss of
infectivity for mice (Lindsay and Dubey, unpublished, 1996). Tachyzoites cryopreserved
Table 2
Attempts to find oocysts in feces of animals infected with N. caninum a
Animal species No. lnoculum Days Bioassayed N. caninum Reference
feces in mice d antibody e
examined
Cat 3 Naturally 21 No Not Cuddon et al. (1992)
(Fells infected tested
domesticus) dog brain
2 Mice infected 30 Yes Yes Dubey and Lindsay
with NC- 1, NC-2, (unpublished, 1996)
NC-3 isolates
1 Brain of an 29 No Yes Dubey and Lindsay
experimentally (unpublished, 1990)
infected cat
3 Tachyzoites NC-I, 30 No Yes Dubey et al. (1990c)
IM, orally
1 Tachyzoites 20 No Yes Dubey and Lindsay
NC-1, SC (1989a)
2 Tissues of a 22 No No Dubey and Lindsay
naturally infected (unpublished, 1988)
COW c
6 Tissues of three 15 No Not Dubey and Lindsay
naturally tested (unpublished, 1989)
infected cows f
2 Tissues of an 17 No No Dubey and Lindsay
experimentally (unpublished, 1989)
infected cow
Dog 1 NC- I, tachyzoites, 30 No Yes Dubey and Lindsay
(Canis oral and SC (1989b)
familiaris)
Raccoons 2 NC- 1 infected 50 Yes Yes Dubey et al. (1993)
( Procyon mouse
lotor) tissues b
Coyotes 2 NC-1, NC-2, NC-3 30 Yes Yes Lindsay et al. (1996c)
(Canis infected mouse
latrans) tissues b
Red-tailed hawk 2 NC-1, NC-2, NC-3 30 Yes Not Baker et al. (I 995)
(Buteo infected mice b tested
jama&ensis)
Turkey vulture 2 NC-I, NC-2, NC-3 30 Yes Not Baker et al. (1995)
(Cathartes aura) infected mice ~ tested
Barn owl 2 NC- 1, NC-2, NC-3 31 Yes Not Baker et al. (1995)
(Tyto alba) infected mice b tested
American crow 3 NC-I, NC-2, NC-3 30 Yes Not Baker et al. (1995)
(Corvus infected mice b tested
brachyrhynchus)
a Oocysts were not found in feces of any of these animals.

b The donor mice had antibodies (IFA > 1:100) to N. caninum.
c The donor cow had a confirmed N. caninum infected fetus (Dubey et al., 1990f).
d Feces were incubated in 2% H2SO 4 on a shaker for 1 week and then fed to BALB/c mice.
c Serum from the carnivore host was tested for N. caninum antibodies in an IFAT.
f Cows had N. caninum antibody (IFAT, l:100).
in liquid nitrogen can retain infectivity for cell cultures; methods used to preserve T.
gondii (Dubey and Beattie, 1988) are suitable for preservation of N. caninum. Tachy-
zoites of NC-1 isolate can divide into two zoites within 8 hours of inoculation (Lindsay
and Dubey, unpublished).
2.4. Host-parasite relationship
Neospora caninum is capable of producing grossly visible necrotic lesions in a few

days, and causes cell death by the active multiplication of tachyzoites. It can produce
severe neuromuscular disease in dogs and cattle and probably other hosts, destroying
large numbers of neural cells, including those of cranial and spinal nerves and affecting
the conductivity of the affected cells (Mayhew et al., 1991; Dubey and de Lahunta,
1993).
Tissue cysts are often not surrounded by a zone of host reaction. Although the length
of time tissue cysts can persist in tissues is not known, they are viable in brains of
experimentally infected mice for at least 1 year (Lindsay et al., 1992). Formation of
granulomas around degenerating tissue cysts and bradyzoites (Dubey et al., 1990b;
Dubey et al., 1992a; Dubey et al., 1996b; Mayhew et al., 1991), suggest that some tissue
cysts rupture, and that the subsequent host reaction causes foci of inflammation.
Administration of exogenous corticosteroids can exacerbate neosporosis (Dubey and
Lindsay, 1990a).
2.5. Antigens
Barta and Dubey (1992) recognized at least 20 proteins ranging from 16 to 80 KDa in
Western blots by using polyclonal serum from a rabbit immunized with live whole N.
caninum tachyzoites. Bjerk~ et al. (1994) characterized four major and several minor N.
caninum antigens. The four major antigens (17, 29, 30 and 37 KDa) were recognized in
sera of both naturally and experimentally infected animals, including cattle and dogs;
these antigens were not recognized by preinoculation sera or sera from uninfected
animals. Using sera raised in rabbits against specific protein bands, the 17 KDa antigen
was found to be associated with rhoptries, while the 39 and 30 KDa antigens were
associated with dense granules, the tubular network and the membranes of the para-
sitophorous vacuole (Bjerk~s et al., 1994). Based on recent studies, it appears that 29
and 30 KDa proteins may be the same (M.C. Jenkins, unpublished, 1996). These
immuno-dominant N. caninum antigens were not recognized in lysates prepared from T.
gondii. Although N. caninum and T. gondii have several common antigens, they do not
share their dominant antigens. For example, homologues of T. gondii B 1, p22 and p30
genes were not detected in the genome of N. caninum by PCR (Brindley et al., 1993).
There is no demonstrable cross protection between the RH strain of T. gondii and Nc-1
isolate of N. caninum in mice (Lindsay et al., 1990a).
Some progress has been made in constructing a N. caninum cDNA library and two
immunoreactive recombinants (Nc 4.1, Clone A and Nc 14.1, Clone B) were identified
using bovine sera from naturally and experimentally infected cattle (Lally et al., 1996a).
Clone A expressed protein of approximately 35 KDa, whereas Clone B expressed
IO J.P. Dubey, D.S. Lindsay / Veterinary Parasitology 67 (1996) 1-59
proteins of approximately 30 KDa. Both of these proteins were recognized by N.

caninum serum antibodies from cows that had aborted due to neosporosis and from
experimentally infected cattle, but not from sera of cows experimentally inoculated with
the related protozoans such as Sarcocystis spp. and T. gondii. These recombinant
proteins could provide a defined antigen for mass screening of sera by ELISA.
Recently, Hemphill and Gottstein (1996) identified and purified a protein (p43) from
N. caninum tachyzoites. Antibodies raised against the p43 protein reacted exclusively
with N. caninum tachyzoite pellicle and not with T. gondii tachyzoites; this appears to
be an important finding because of the possible involvement of p43 in host cell adhesion
and invasion.
Barber et al. (1995) compared antigenic patterns of 2 (NC-1 and NC-Liv) isolates of
N. caninum and found generally similar bands in the molecular weight range of 16 to 80
KDa; they reported a 28 KDa band in NC-Liv not found in the NC-I tachyzoite
homogenate, however, the results were not conclusive.
Compared with antigen composition of tachyzoites of N. caninum, little is known
about the antigens of tissue cysts. Immunohistochemical studies have demonstrated
occasional cross reactivity between tissue cysts of T. gondii and N. caninum using
polyclonal sera (Dubey et al., 1990d; Ruehlmann et al., 1995). McAllister et al. (1996c)
have demonstrated that a bradyzoite-specific recombinant antigen (BAG5) from T.
gondii will cross react with N. caninum bradyzoites but not with tachyzoites.
2.6. Molecular biology
There is a growing interest in studying N. caninum at the molecular level. Molecular

characterizations of N. caninum have focused mostly on taxonomy and development of
PCR diagnostic tests. Available information on N. caninum isolates is summarized in
Table 3. The taxonomic position of N. caninum is uncertain because its life cycle has
not been defined. Analysis of the small subunit ribosomal RNA (ssrRNA) sequence of
N. caninum and T. gondii showed consistent four nucleotide differences between these
two parasites (Marsh et al., 1995). Similar comparisons between bovine and canine
Neospora isolates under identical conditions showed no nucleotide differences (Marsh et
al., 1995). Although differences were noted between sequences derived from the same
strain of N. caninum (NC-1) technical problems associated with reading the nucleotide
sequencing gels may account for some of the differences between the sequences in
GenBank (Marsh et al., 1995). Because of the close sequence homology between the
16s-like rRNA genes of T. gondii and N. caninum, it has been suggested that N.
caninum should be placed in the genus Toxoplasma (Ellis et al., 1994; Holmdahl et al.,
1994). However, a recent comparison using the random amplified polymorphic DNA
(RAPD) PCR, Guo and Johnson (1995) found significant DNA polymorphisms among
the genera Neospora, Toxoplasma and Sarcocystis and concluded that N. caninum was
an independent species. They did not find an especially close relationship between
Toxoplasma and Neospora. Brindley et al. (1993) had earlier reached the same
conclusion that homologues of the three dominant T. gondii B1, p22 and p30 genes
were absent in N. caninum. It is noteworthy that canine isolates of N. caninum from
USA and England had identical homology not only in the ssrRNA, but also in the ITS-1
Table 3
Summary of recognized isolates of Neospora caninum
Isolate GenBank Date inoculated Source Region Reference
designation accession no. in culture a
NC-I U17346 2 February 1988 Tissues of two Pennsylvania, Dubey et al. (1988b)
U03069 congenitally USA
L24380 infected dogs
U16160
X84238
U31542
NC-2 9 June 1988 Muscle biopsy Virginia, Hay et al. (1990)
of a dog USA
NC-3 U25044 7 August 1988 Brain and spinal Wisconsin, Cuddon et al. (1992)
cord of a dog USA
NC-Liv U16159 13 August 1993 Brain of a dog Liverpool, Barber et al. (1993);
L49389 England Barber et al. (1995)
NC-SweBI 9 March 1995 Brain of a Uppsala, Stenlund et al. (1996)
stillborn calf Sweden
BPA-I U17345 13 June 1991 Bovine fetal California, Conrad et al. (1993a)
brain USA
BPA-2 U25043 20 September 1991 Bovine fetal California, Conrad et al. (1993a)
brain USA
BPA-3 U25043 8 January 1992 Brain of a California, Marsh et al. (1995);
congenitally USA Barr et al. (1993)
infected calf
BPA-4 U25043 13 February 1992 Congenitally California, Marsh et al. (1995);
infected calf USA Barr et al. (1993)
JPAI 1 February 1996 Congenitally Ibaraki, Yamane et al., 1996
infected calf Japan
a Based on personal communication with authors.
region (Barber et al., 1995) which is variable and shows considerable divergence from
Toxoplasma (Holmdahl and Mattsson, 1996).
The information derived from genomic DNA of N. caninum has potential for
diagnosis of neosporosis (Kaufmann et al., 1996). Information on PCR utilizing different
genes and techniques is summarized in Table 4. A potential disadvantage which might
apply to PCR methods that use 16s-like RNA genes is the possibility of cross reaction
with unknown organisms because this is a highly conserved gene. However, most
researchers tested specificity against Sarcocystis and Toxoplasma infected material. The
possibility of cross reaction could also be a problem with the PCR method of Lally et al.
(1996b) because they used a conserved eukaryotic protein gene. However, the degree of
conservation is not nearly as high as for rRNA genes. They have tried to further
minimize the risk of cross-reactivity by choosing primers directed towards variable
regions of the gene, and from outside the coding region where the sequence is less
conserved. Although Lally et al. (1996b) and Yamage et al. (1996) were able to detect
N. caninum DNA in infected brain tissue and Ho et al. (1996) were able to detect N.
caninum in infected amniotic fluid, the problem of detecting N. caninum DNA in
autolyzed tissues has not yet been addressed. The PCR addressing the sequence of ITS- 1
12 J.P. Dubey, D.S. Lindsay / Veterinary Parasitology 67 (1996) 1-59
Table 4
Use of PCR-tests for the diagnosis of neosporosis
Reference Method Sequence Sensitivity and remarks
Holmdahl and PCR ITS- 1 (Internal transcribed Five tachyzoites from cell
Mattsson (1996) spacer) between 5.8S and 16S-like culture-tachyzoite suspension
rRNA genes
Ho et al. (1996) PCR + SSrRNA + hybridization with Five tachyzoites, tested in 100
hybridization Neospora-specific ,ttl of bovine and monkey
oligonucleotide probe whole blood or amniotic fluid.
The process requires two steps,
PCR followed by hybridization
Lally et al, (1996b) Two stage 14-3-3 protein gene 25 taehyzoites in cell culture
' nested' PCR suspensions, also bradyzoites
in mouse brain. This procedure
requires two stages of PCR.
This procedure is not as time
consuming as PCR followed
by hybridization
Yamage et al. (1996) PCR pNC-5 Neospora specific genomic One tachyzoite in cell culture
DNA sequence of unknown and tachyzoites and
function. bradyzoites in brain tissue
Payne and Ellis PCR ITS-1 between 5.8s and Sensitivity not assessed
(1996) 18s rRNA genes.
of N. caninum (Holmdahi and Mattsson, 1996; Payne and Ellis, 1996) has proved
specific as well as sensitive. The ITS-1 sequence is highly repetitive and has shown to
be conserved within species but variable between species. By the PCR developed by
Holmdahl and Mattsson (1996) it was possible to detect N. caninum in brain as well as
liver and lung of experimentally infected animals. This PCR was also successfully
applied for the detection of N. caninum in body fluids and tissues of experimentally
infected sheep (CSF, amniotic fluid, buffy coat, cotyledon, etc.) (Holmdahl, personal
communication, 1996) By the PCR developed by Payne and Ellis (1996) it was possible
to detect N. caninum in formalin fixed tissues (Ellis, personal communication, 1996).
2.7. Diagnosis
Although neosporosis may resemble toxoplasmosis clinically, T. gondii and N.

caninum can be distinguished antigenically and sometimes structurally (Dubey et al.,
1988a). Neospora caninum tachyzoites are identical to T. gondii tachyzoites under the
light microscope but can be distinguished using transmission electron microscope based
on the appearance of their rhoptries (Dubey et al., 1988a; Lindsay et al., 1993).
Rhoptries in N. caninum tachyzoites are electron-dense, whereas those in T. gondii
tachyzoites are honey-combed. The number of rhoptries and the arrangement of mi-
cronemes alone cannot be used to distinguish N. caninum from T. gondii because their
number and arrangements vary a great deal. Well-developed tissue cysts of N. caninum
J.P. Dubey, D.S. Lindsay/ Veterinary Parasitology67 (1996) 1-59 13
can be distinguished from T. gondii through light microscopy (Dubey et al., 1988a).
Neospora caninum tissue cysts occur in neural tissues, and the tissue cyst wall is up to 4
/zm thick, whereas T. gondii tissue cysts may be found in many organs and the tissue
cyst wall is always < 1 p.m thick.
Serologic examination can aid diagnosis. Both the indirect fluorescent antibody test
(IFAT) and enzyme linked immunosorbent assay (ELISA) have been used to detect N.
caninum antibodies. Although N. caninum shares several antigens with T. gondii, the
IFAT seems to be specific. Several factors such as the conjugate, buffers, and the pattern
of fluorescence are important when evaluating specificity of the serologic test. The IFAT
was first developed to detect N. caninum antibodies in dogs (Dubey et al., 1988b).
Cross reactions were not seen with T. gondii at a serum dilution of 1:100 when
compared in the IFAT for both organisms. Dubey et al. (1996a) examined, in detail,
serologic responses of cattle, sheep, goats, pigs, rabbits, mice and rats to experimental
N. caninum infection and found little or no cross reactivity with T, gondii using the
IFAT, modified agglutination test (MAT) or the classical Sabin-Feldman dye test for T.
gondii antibodies in these animals. Sera with high N. caninum antibodies did not react
in the T. gondii in MAT at 1:25 dilution and in the dye test at 1:16 dilution (Dubey et
al., 1996a). Cross reactive antibodies were found in two raccoons fed N. caninum
(Dubey et al., 1993). One raccoon had T. gondii titer in the DT of 1:10 and the other
had transitory titers of 1:10, 1:80, 1:20, 1:20, and 1:10 on 11, 17, 23, 30 and 38 days
after inoculation (DAI), respectively. Dairy goats inoculated with N. caninum developed
slight cross reactivity to T. gondii in the IFAT, but not in the MAT (Dubey et al.,
1996a), In another experiment, rabbits inoculated with Sarcocystis neurona, S. muds, S.
cruzi, or Hammondia hammondi did not develop antibody titers to N. caninum in the
IFAT (Dubey et al., 1996a). Some serologic cross reactivity has been reported among
Babesia gibsoni, Babesia canis, T. gondii and N. caninum (Yamane et al., 1993). Sera
from two dogs experimentally infected with B. gibsoni reacted to N. caninum and T.
gondii antigens, but sera from dogs experimentally infected with N. caninum did not
have B. gibsoni antibodies. Sera from dogs experimentally infected with T. gondii did
not react with N. caninum in the IFAT (Yamane et al., 1993). However, cross reactivity
was not found between the MAT for T. gondii and the IFAT for N. caninum. In all
parasitologically confirmed cases of canine neosporosis had titers of >_ 1:200 in IFAT
(Lindsay and Dubey, unpublished). In all 20 clinical cases from which serum was tested
in the United Kingdom IFAT titers were > 1:1280 where as such titers were found only
in 1.2% in healthy dogs (Barber and Trees, 1996). Additionally, Trees et al. (1993) did
not find any association between the presence or absence of anti-T, gondii antibodies by
the dye test and anti-N, caninum antibodies by IFAT in naturally infected dogs.
There is more cross reactivity between T. gondii and N. caninum in dogs using a
crude N. caninum extract as antigen in ELISA than in a conventional IFAT (Dubey and
Lindsay, 1993). BjiSrkman et al. (1994a) overcame some of these cross reactivity
problems by combining soluble extracts of N. caninum with immunostimulating com-
plexes (iscoms). The electrophoretic analysis of the N. caninum iscoms antigen indi-
cated that the major N. caninum proteins in the mixture had molecular weights of 17 to
18 and 30 to 45 KDA. They found good agreement between their iscom-ELISA and the
IFAT. The ELISA when compared with the IFAT has the advantage that it is suitable for
14 J.F. Dubey, D.S. Lind,~ay/ Veterinary Parasitology 67 (1996) 1-59
automation. The use of purified N. caninum specific proteins as antigen might improve
the specificity of the ELISA. Lally et al. (1996a) have identified two recombinant N.
caninum proteins that appear to be promising for an N. caninum-specific ELISA.
Celt culture and mouse inoculations can also be used to recover N. caninum from
animal tissues (Dubey et al., 1988b). The success of isolation depends on the number of
organisms present and the state of autolysis. Conrad et al. (1993a), using cell culture,
isolated N. caninum from only two of more than 100 bovine fetuses suspected to have
been aborted because of N. caninum. This monumental effort illustrates the difficulty of
diagnosing N. caninum abortions. Bryan et al. (1994) recovered N. caninum in mice
inoculated with neural tissue from a calf in Canada; this result is of interest because the
bovine tissue had been frozen at - 5 2 C for about 4 months before inoculation into
mice.
Neospora caninum can be distinguished in sections immunohistochemically (IHC)
using anti-N, caninum serum (Lindsay and Dubey, 1989c). Most reports of neosporosis
in animals are based on the specific staining of N. caninum using polyclonal serum from
rabbits immunized with cell culture-derived tachyzoites. Occasionally, N. caninum
reacts weakly with T. gondii antiserum. However, it must be remembered that none of
the immunohistochemical reagents are 100% uniform, which may result in minor
differences in specificity observed in various laboratories. Additionally, there can be
considerable variation in reactivity of antiserum depending on the source of rabbits used,
the type of antigens, stage of the parasite used to immunize rabbits, and fixation of the
parasite antigens. Other technical aspects such as incubation times, trypsin or pepsin
treatment of tissues, or lack of enzyme treatment of tissues can influence the results of
these immunohistochemical tests. Occasionally, some blocks of tissues taken from the
same animal and fixed identically will react differently in immunohistochemical tests.
Therefore, diagnosis should not be based solely on the results of a single immunohisto-
logical examination. A monoclonal antibody-based system specific for N. caninum
tachyzoites and tissue cysts has been developed that reacts in immunohistochemical tests
(Cole et al., 1993, Cole et al., 1994).
2.8. Experimental treatment
Progress is being made in determining the susceptibility of tachyzoites of N. caninum

to chemotherapeutic agents and baseline data is available for a variety ( > 40) of agents
(Lindsay and Dubey, 1989b; Lindsay et al., 1994, Lindsay et al., 1996a). Cell culture-
based assays for identifying effective agents aid in the discovery of effective treatments,
because a large number of agents can be examined efficiently over a short period. Cell
culture-based assays also lend themselves to drug structure-drug activity studies, which
can lead to the logical design of better agents.
The efficacy of treatment with several sulfonamides dihydrofolate
reductase/thymidylate synthase (DHFR/TS), inhibitors, ionophorous antibiotics
(lasalocid, maduramicin, monensin, narasin, and salinomycin), macrolides (azithromy-
cin, clarithromycin, and erythromycin) and tetracycline (doxycycline and minocycline)
antibiotics and lincosamide antibiotics have been evaluated in cell culture and with the
exception of the sulfonamides all have activity (Lindsay et al., 1994, 1996d). Arprinocid,
Table 5
Efficacy for combinations of sulfonamides and dihydrofolate reductase/thymidylate synthase ( D H F R / T S )
inhibitors against tachyzoites of the NC-1 isolate of Neospora caninum in human fibroblast cell cultures
DI-IFR/TS inhibitors b
Sulfonamide a Dia Met Orm Pyr Tri
Sulfadiazine 100 12 20 100 100
Sulfadimethoxine 100 ND 41 91 100
Sulfamerazine 100 ND 84 61 96
Sulfamethazine 100 ND 100 93 100
Sulfaquinoxaline 96 10 42 41 0
Sulfathiazole 1O0 10 1O0 1O0 1O0
a All sulfonamides were given at 10 /.~g m l - t.

b Dia: 1.0 /.~g m l - l diaveridine; met: 10.0 p,g m l - t methotrexate; orm: 0.1 p,g m l - ' ormetoprim; pyr: 0.01
~ g m l - z pyrimethamine; tri: 1.0/~g m l - ~ trimethoprim.
Table 6
Efficacy b of dihydrofolate reductase/thymidylate synthase (DHFR/TS) inhibitors against pyrimethamine
resistant mutants of Neospora caninum
DHFR/TS DoSe a Control Pyr R- 1 PyrR-2
inhibitor
Piritrexim 0.OO01 40 + 16 30
0.001 loo 18 100P
Pyrimethamine 0.01 0 ND ND
0.025 98 ND ND
0.05 100P ND ND
0.075 100 ND ND
0.1 loo ND 0
0.5 ND ND 60
0.75 ND ND 97
1.0 IOO 0 IOO
2.5 ND 100P ND
5.0 ND IOO ND
Ormetoprim 0.1 4 ND ND
0.25 10 ND ND
0,75 19 ND ND
1.0 100P 0 0
10.0 loo 0 95
Diaveridine 1.0 0 ND ND
2.5 100P ND ND
7.5 100 ND ND
10.0 IOO 0 73
Trimethoprim 1.0 0 ND ND
2.5 100P ND ND
7.5 IOO ND ND
10.0 100 10 34
a Dose is in /.tg m l - i.
b Percent efficacy; ND: not determined; P: tachyzoites present but no lesions observed in cell cultures.
nifrofurazone, robenidine, decoquinate, and diclazusil are also effective in cell culture.
Amprolium, metronidazole, paromomycin, and roxarsone have little to no activity
against N. caninum tachyzoites (mLindsay et al., 1994). Synergism has been demon-
strated for combinations of pyrimethamine, ormetoprim, trimethoprim, and diaveridine
with sulfonamides against tachyzoites of N. caninum in cell cultures (Lindsay et al.,
1996a) (Table 5). Methotrexate does not demonstrate improved efficacy when combined
with sulfonamides.
Two mutants of N. caninum have been produced that are resistant to pyrimethamine
(Lindsay et al., 1996a). One mutant was selected using low levels of pyrimethamine and
designated the PyrR-1 mutant. The other was produced by chemical mutagenesis with
N-methyl-N-nitro-N-nitrosoguanidine and designated the PyrR-2 mutant. Both mutants
were resistant to other DHFR/TS inhibitors (Table 6). Both mutants remained resistant
to pyrimethamine in the absence of continuous exposure to the agent indicating that the
induced resistance was stable. Synergism was demonstrated for combinations of
DHFR/TS inhibitors and sulfonamides against these pyrimethamine resistant mutants
(Lindsay et al., 1996a).
Few compounds have been evaluated experimentally for their effects against N.
caninum in vivo. Amprolium did not prevent the development of neosporosis and deaths
in mice treated with four or 20 mg per mouse daily in the drinking water 3 days after
inoculation (DAI) of tachyzoites (Lindsay and Dubey, 1990a). Sulfadiazine given in the
drinking water at 4 mg per mouse, 3 DAI for 14 days protected 90% of mice (Lindsay
and Dubey, 1990a). Sulfadiazine given in the drinking water at 2 mg per mouse 3 DAI
for 28 days protected 50% of the mice. Neither amprolium nor sulfadiazine given at 4
mg per mouse in the drinking water was effective if treatment was initiated 7 days after
tachyzoites were given (Lindsay and Dubey, 1990a).
3. Neosporosis in dogs
3.1. Seroprevalence in the general population
Only limited information is available concerning prevalence of N. caninum infection

in dogs. In four convenience surveys of dogs brought to veterinary clinics in Kansas,
USA; Liverpool, UK; North Mymms, UK; and Uppsala, Sweden; seroprevalence was
0.25 to 16.6%. Using the IFAT, five of 229 dogs from Kansas had antibodies in serum
dilutions of 1:400 (two dogs), 1:200 (two dogs) and 1:100 (one dog) (Lindsay et al.,
1990b). The prevalence of T. gondii antibodies in the same dogs was 25% and three of
the five dogs with N. caninum antibodies also had T. gondii antibodies. Of the 163 dogs
surveyed in Liverpool, 50 had titers of 1:50, eight had titers of 1:200, nine had titers of
1:800 and four had titers of 1:3200 (Trees et al., 1993). Thus, 17% were positive at a
titer of 1:50 and 13% were positive at a titer of 1:200. Of the 398 dogs from Uppsala,
tested by IFAT and ELISA, one had an IFAT titer of 1:80 and two were positive by
iscom-ELISA (Bj~rkman et al., 1994b). There was no cross reactivity between N.
caninum and T. gondii in both surveys. Of 104 dogs from North Mymms, N. caninum
antibodies were found in a 1:50 serum dilution of six of 104 (6%) dogs (Lathe, 1994).
3.2. Clinical signs
The most severe cases of neosporosis occur in young, congenitally infected pups.
Young dogs develop hind limb paresis that develops into a progressive paralysis.
Neurologic signs are dependent on the site parasitized (Jackson et al., 1995). The hind
limbs are more severely affected than the front limbs and often in rigid hyperextension
(Cuddon et al., 1992; Dubey, 1993a; Barber and Trees, 1996; Barber et al., 1996). Other
dysfunctions which occur include difficulty in swallowing, paralysis of the jaw (Hay et
al., 1990), muscle flaccidity, muscle atrophy and even heart failure (Odin and Dubey,
1993). Dogs with hind limb paralysis may be alert and survive for months.
The cause of the hind limb hyperextension is not known, but is most likely due to a
combination of upper motor neuron paralysis and myositis which results in rapidly
progressive fibrous contracture of the muscles that may cause fixation of joints. The
disease may be localized or generalized and virtually all organs may be involved
including the skin (Dubey et al., 1988a, Dubey et al., 1995). Dermatitis may be severe
involving enormous numbers of N. caninum. Dogs of any age may be affected. Fatal
neosporosis has been reported in 8 to 15-year-old dogs (Dubey et al., 1988a, Dubey et
al., 1995; Hoskins et al., 1991).
Subclinically infected bitches can transmit the parasite to their fetuses, and successive
litters from the same bitch may be born infected (Dubey et al., 1990d). Whether there is
breed predisposition and differential sex susceptibility to neosporosis in dogs is not
known. Most described cases have been in Labrador retrievers, Boxers, Greyhounds,
Golden retrievers, and Basset hounds (Table 7).
3.3. Diagnosis
For antemortem diagnosis, clinical signs may be helpful. Ascending paralysis in

young dogs, particularly if several littermates are affected, should arouse suspicion of
neosporosis. Hematological values are usually not altered. There may be increased levels
of serum enzymes associated with necrosis of myocytes and occasionally hepatocytes.
A serologic examination can aid diagnosis. The IFAT is currently the most com-
monly used diagnostic test and reagents are commercially available (VMRD, Pullman,
Washington). In our experience, all clinical cases of dogs with proven neosporosis had
an IFAT titer of from >- 1:200 to often more than 1:1600. Detection of antibodies in the
cerebrospinal fluid (CSF) is diagnostic. For all practical purposes, the IFAT is specific.
The cross reactivity seen in some experimentally infected dogs by Yamane et al. (1993)
is not a problem because there is very little cross reactivity between T. gondii and N.
caninum in naturally infected dogs (Trees et al., 1993). Obviously, dogs could be
infected with both N. caninum and T. gondii (Dubey et al., 1995). Antibodies to N.
caninum can be detected by an iscom-ELISA (BjSrkman et al. (1994a).
The detection of N. caninum in biopsy tissue or cytological preparation can confirm
diagnosis (Fig. 5). Neospora caninum has been detected in biopsy of muscles (Hay et
al., 1990), lung aspirates (Greig et al., 1995), and in dermal pustular exudate (Dubey et
al., 1988a; Dubey et al., 1995). Immunohistochemical staining is needed to exclude
toxoplasmosis because tachyzoites of N. caninum are indistinguishable from T. gondii
Table 7
Summary of reports of neosporosis in single dogs
Country Reference No. of Breed Age Clinical signs
dogs
2
Australia Munday et al. (1990) 4 2 Greyhounds, 2 unknown < 3 months, < 3 months, Neurologic in 3 and
> 6 months, < 3 months sudden death in 1
Gasser et al. (1993) I Boxer 6 months Neurologic
Belgium Poncelet et al. (1990a,b) 5 3 Boxers, I Doberman, 2 months, 2 months, 2.5 months, Neurologic in all 5
1 Labrador 1 month, 1 month
Canada Odin and Dubey (1993) I Bullmastiff 10 months Sudden death, myocarditis
Cochrane and Dubey (1993) I Golden retriever 12 weeks Neurologic
Costa Rica Morales et al. (1995) I Cocker spaniel 2 years Neurologic
England Dubey et al. (1990c) 1 Shetland sheep dog 9 months Neurologic
Knowler and Wheeler 3 2 Boxers, 1 Labrador 5 weeks, 18 months, 15 weeks Neurologic and neuromuscular
(1995) retriever in both
Finland Rudb~ick et al. (1991) 1 1 Great dane 8 months Neurologic, polyuria
France Poncelet et al. (1990b) 1 Bleu de picardie 1 month Neurologic
France Fritz et al. (1996) 1 Siberian husky 6 years Dermal
Germany Bur "ldtardt et al. (1992) 2 1 Boxer, 1 Labrador retriever 2 months, 5 months Neurologic in both
Hungary Sr6ter et al. (1992) I Basset hound 2 months Neurologic
Ireland Sheahan et al. (1993) 5 1 Boxer, 4 Greyhounds 6 months, 7 weeks, 9 weeks, Neurologic in all 5
10 weeks, 6 months
Japan Umemura et ak (1992) l I Shetland sheep dog 5 months Neurologic
South Jardine and Dubey (1992) 3 t Great dane 12 weeks Neurologic
Africa 1 Labrador retriever 6 months Neurologic
1 Scottish terrier 2 weeks Neurologic
Spain Pumarola et al. (1996) Napolitan mastiff 4 months Neurologic
Sweden Hilali et al. (1986) Greyhound 4 months Neurologic,
Uggla et al. (1989a) Boxer 10 weeks Neurotogic
Uggla et al. (1989b) Riesenschnauzer 4 years Neuromuscular
Switzerland Wolf et al. (1991) 5 Boxers 1 Labrador retriever 'Pups' Neuromuscular in all
1 Golden retriever 1 Bemhardiner
1 Irish Wolfhound
USA Braund et al. (1988) a 2 1 Bloodhound 3.5 years Neuromuscular
1 Borozi 6 years Neuromuscular
Dubey et al. (1988a) 10 1 Shetland sheepdog 8 months Neurologic
3 Basset hounds 8 months, 5 years, 5 months Neurologic in all 3
1 Bullmastiff 3 months Neurologic
1 Collie 4 years Neurologic
e~
1 Golden retriever 6 months Neurologic and myocarditis
1 Mixed-breed 15 years Dermal
2 Poodles 2 years Neurologic
6 weeks Polymyositis
Hay et al. (1990) Labrador retriever 12 weeks Neurologic
Hoskins et al. (1991) Basset hound 10 years Neurologic c,
Ruehlmann et al. (1995) Vizsla 3 years Neurologic

Jackson et al. (1995) Golden retriever 3.5 years Neurologic
Dubey et al. (1995) Golden retriever 12 years Dermal
a Retrospective identification.
7"
20 J.P. Dubey. D.S. Lindsay / Veterinary Parasitology 67 (1996) 1-59
Fig. 5. Neospora caninum tachyzoites in skin of naturally infected dogs. Bar: l0/xm and applies to A to C. A.
Smear of a dermal ulcer. Note numerous single (arrowheads), binucleate (double arrowhead), and nearly
divided tachyzoites (arrow). Methanol fixed, Giemsa stain. B. Section stained with H&E stain. Note single
(arrowhead), pair (double arrowhead) and large groups (arrows) of tachyzoites among pustular inflammatory
cells, mostly neutrophils. C. Section stained with N. caninum-specific antibodies. Note several individual
(arrowheads) and groups (arrow) of tachyzoites.
by light microscopy. Tachyzoites should be looked for in C S F in cases o f hind limb

paralysis.
P o s t m o r t e m diagnosis can be based on demonstrating the parasites in lesions o f
affected dogs. Gross lesions o f neosporosis reported were necrosis in the central nervous
system (CNS) and liver (Dubey et al., 1988a), granulomas (up to 1 cm in diameter) in
visceral tissues (Dubey et al., 1988a), yellowish white streaks in muscles, particularly in
diaphragm (Dubey et al., 1988b; Dubey and Lindsay, 1990a) cerebellar atrophy (Bjerk~s
and Presthus, 1989; Jackson et al., 1995) and ulcerative dermatitis (Dubey et al., 1988a,
Dubey et al., 1995). Immunohistochemical tests using specific antibodies will confirm
the diagnosis (Fig. 5C). The parasites most likely confused with N. caninum are T.
gondii and Sarcocystis canis (Dubey, 1993a). Sarcocystis canis divides by en-
dopolygeny into many organisms, whereas T. gondii and N. caninum divide into two
organisms by endodyogeny.
3.4. Reports of clinical neosporosis
Clinical neosporosis has been reported from dogs in Australia, Belgium, Canada,
Costa Rica, Denmark, UK, Finland, France, Germany, Hungary, Ireland, Japan, Nether-
lands, Norway, South Africa, Spain, Sweden, Switzerland, and USA.
Table 7 summarizes information from other documented cases where only a single
dog was presented for examination or the complete litter history was not known. It is
important to point out that several of these dogs were more than 1 year of age. It is not
known if these cases in older dogs represent relapses of congenital infections or if they
represent primary infections.
Neosporosis involving littermates will be discussed for each of the 17 litters reported.
Litters 1 to 3. Three successive infected litters (Litters 1-3) consisting of a total
seven affected dogs, were produced by a Boxer bitch in Norway (Bjerk~ et al., 1984;
Bjerk~s and Dubey, 1991). Six of the pups developed ataxia and paresis at 2 to 6 months
of age and were euthanized. Encephalomyelitis and myositis associated with N. caninum
stages were observed in the tissues of dogs that were examined at necropsy.
Litter 4 to 6. Four Labrador retriever pups from a litter of eight (Litter 4) developed
asymmetric paraparesis after 4 weeks of age in Pennsylvania, USA (Cummings et al.,
1988). The hind limb weakness progressed to tetraplegia in three of the four. Cervical
weakness, inability to prehend food, and dysphagia also developed. These three pups
died soon after development of these signs and the fourth was euthanized. Lesions in the
pups consisted of meningoencephalomyelitis and polyradiculoneuritis associated with N.
caninum. The bitch was rebred and gave birth to seven pups (Litter 5). These pups
developed normally for 5 to 6 weeks then developed hind limb paresis (Dubey et al.,
1988b). Three pups were killed by the owner. Three were examined at necropsy and one
mildly affected pup survived with minimal ataxia. A second bitch from this household
gave birth to a litter of seven pups (Litter 6). One pup died at 3 weeks and was not
examined. Three pups developed hind limb paresis at 8 weeks of age. Two were
euthanized at 12 weeks and were examined, the third dog was euthanized by the owner
and was not necropsied. The remaining three dogs remained normal and were sold.
Lesions of encephalomyelitis, polyradiculoneuritis, and polymyositis were observed in
the tissues of dogs in Litters 9 and 10. Neospora caninum was isolated from the tissues
of these dogs in cell cultures and laboratory animals (Dubey et ai., 1988b).
Litters 7 to 10. All four litters were owned by the same person in Ohio, USA, and
were German shorthaired pointers (Dubey et al., 1990d). This was a retrospective study
of cases that occurred in 1957. In Litter 7, eight of nine pups developed limb paralysis
and died before 6 months of age. The surviving pup was a female and was the dam of
Litters 9 and 10. The dam from Litter 7 was bred to a different sire and gave birth to 11
pups. One was killed because it was a runt and eight others developed hind limb
paralysis and were euthanized. Two males survived and one eventually developed
lameness in the left hind limb and the other remained normal. The surviving female of
Litter 7 was bred to the sire of Litter 9. Nine pups were born and seven died before
weaning and were not examined. The remaining two dogs from Litter 9 developed ataxia
at 2 weeks of age. One eventually developed hind limb paralysis and both were
euthanized. This bitch gave birth to ten pups in Litter 10. The sire was the surviving
male of Litter 8. One pup died at 2 days of age. One pup developed hind limb paresis at
3 weeks of age and the remaining 8 pups developed paralysis in one or both hind limbs
by 2 months of age. They remained alert. Three of these dogs died at 84, 107, and 240
days of age and another was euthanized at 165 days of age. Encephalomyelitis and
myositis associated with N. caninum were observed in the tissues of dogs that were
examined at necropsy.
Litters 11 and 12. A Bloodhound bitch in Suffolk, UK gave birth to a litter of three
puppies (Litter 11) which developed progressive neurological signs (Mayhew et al.,
1991). The most severely affected pup developed signs at 5 weeks of age that progressed
to rigid fixed extension of both hind limbs in the next 4 weeks. A second pup developed
hind limb ataxia at 12 weeks of age and the third remained normal. The most severe!y
affected pup was euthanized at 17 weeks of age. Lesions of encephalomyelitis and
myositis were detected in its tissues. The moderately affected pup was treated with
pyrimethamine and trimethoprim plus sulfadiazine for 4 weeks and its signs resolved.
The dam was rebred and gave birth to nine pups, 11 months (Litter 12) later. The dam
had been given anti-Neospora treatment before breeding and at weekly intervals during
pregnancy. Three of the nine pups developed signs consistent with neosporosis.
Litter 13. Four male English Springer spaniel pups from a litter of five in Wisconsin,
USA developed progressive paraparesis in the first 10 weeks of life (Cuddon et al.,
1992). The single female pup in this litter was unaffected. Two of the four male pups
were euthanized and were not examined. One of the remaining pups developed
asymmetric hind limb extensor rigidity. Lesions in the dogs consisted of en-
cephalomyelitis, polyradiculitis and polymyositis. Neospora caninum was isolated in
laboratory animals and cell cultures inoculated with tissues from one of these dogs.
Litter 14. Three of 11 Labrador retriever pups in a litter (Litter 14) from Natal South
Coast, South Africa, developed a progressive neuromuscular disease affecting the hind
limbs beginning at about 4 weeks of age (Jacobson and Jardine, 1993). It progressed to
paralysis and rigid hyperextension of the hind limbs in all three pups when they were 9
weeks of age. One pup was treated with clindamycin and prednisolone with no
improvement noted. All three pups were euthanized. Lesions in the pups consisted of
encephalomyelitis and myositis.
Litter 15. A 7-year-old Labrador Retriever bitch in the Netherlands gave birth to nine
live and one stillborn pup (Wouda et al., 1993), Two pups (Pups 1 and 2) developed
diarrhea on day 25 after birth, The owner had noticed that Pup 1 was dragging one leg a
day earlier; this pup (No. 1) died on Day 25. PUp 2 recovered from diarrhea, but
developed a paralyzed leg when 29 days old and was euthanized on Day 34 because of
paraplegia. Pup 3 developed paresis of the hind limbs on Day 38 and was euthanized on
Day 47 because of poor prognosis. Pup four became ataxic at 5 months of age and was
euthanized. The remaining five pups remained clinically normal. Three pups (2 to 4)
were necropsied. All three pups had encephalomyelitis associated with histologically
proven N. caninum infection. The neurologic deficits in Pups 2 and 3 were considered
to be due to polyradiculoneuritis.
Litter 16. Two of ten pups from a litter of Boxers from Liverpool, UK developed
neurologic signs when 4 weeks old (Barber et al., 1993, Barber et al., 1995). One pup
was followed clinically. This pup had difficulty squatting at 4 weeks of age and
developed paresis in all limbs starting with hind limbs. The pup had an N. caninum
IFAT titer of 1:3200 and titers of 1:200 in the dam and two littermates that had no
clinical signs. The pup was euthanized 3 days after treatment with trimethoprim and
sulfadiazine (Tribrissen , 30 mg kg-1 daily) because it developed dysphagia and
dyspnoea. Neospora caninum was found in histologic sections of brain and skeletal
muscles and was isolated (NC-Liv strain) in cell culture (Barber et al., 1993, Barber et
al., 1995).
Litter 17. A Labrador Retriever bitch gave birth to eight pups in Denmark (Flagstad
et al., 1995). Three pups developed gait abnormalities. One ataxic pup was euthanized at
12 weeks of age without clinicopathologic examination. Another pup developed ataxia,
was evaluated clinically, and euthanized. At complete necropsy was performed. Predom-
inant lesions were encephalomyelitis and myositis and were associated with N. caninum.
The diagnosis was confirmed immunohistochemically. The third pup developed stiff
pelvic limb at 16 weeks of age, but otherwise remained normal for the next 6 months.
The bitch and the remaining five pups were climically normal. Antibodies (IFAT,
> 1:600) to N. caninum were found in sera of all seven pups and the bitch.
Recently, Barber and Trees (1996) reported 27 cases of neosporosis in dogs from the
UK and Europe. Most of these were isolated cases, although some involved littermates.
Breeds involved were Labrador Retrievers (eight), Boxers (seven), Greyhounds (two)
and one each of Bloodhound, Border Collie, Bull Mastiff, Cavalier King Charles
Spaniel, Flat-coated Retriever, Golden Retriever, Hamilton StiSrane, Irish Wolf Hound,
Luzcher and West Highland White Terrier. They were 2-days to 7-years-old. Twenty-one
cases were initially presented with some gait abnormality. Other clinical signs were:
muscle atraphy (14/18), rigid hyperextension (10/19), paralysis (11/21), head tilt
(4/17), dysphagia (4/16) and seizer (1/20). Twenty dogs tested had IFAT titers of
> 1:800. Seveteen dogs were necropsied. Neospora caninum was identified in hema-
toxylin and eosin stained sections of tissues of 15 dogs, by IHC alone in one dog and
ultrastructurally in one dog. In two dogs neosporosis was diagnosed in littermates. In
another case, neosporosis had been diagnosed in a previous litter.
3.5. Treatment
Treatment has been successful in some dogs with early neosporosis-induced limb
weakness. The response to treatment is dependent on the stage of disease that a dog is in
when treatment is initiated. Sulfonamides and/or pyrimethamine, and clindamycin have
partial success in treating canine neosporosis (Barber and Trees, 1996). A combination
of trimethoprim and sulfadiazine (Tribrissen , Coopers Pitman-Moore) at the standard
combined dose of 15 mg kg-1 twice daily and pyrimethamine (Daraprim , Wellcome)
at 1 mg kg-~ daily, all for 4 weeks reversed N. caninum associated paralysis in some
dogs (Mayhew et ai., 1991; McGlennon et al., 1990). Treatment with clindamycin
phosphate (Cleocin phosphate, 150 mg IM every 6 h) for 24 days followed by 3 days of
clindamycin hydrochloride (Cleocin tablets, UpJohn Co., 225 mg orally every 6 h) for 3
days, proved useful in treating protozoai myositis in a 6-year-old male Weimaraner dog
initially diagnosed as toxoplasmosis (Greene et al., 1985), but retrospectively considered
to be neosporosis. However, oral clindamycin (10 mg kg-~ every 8 h) was not effective
in a Labrador retriever pup with rigid hind limb extension and paralysis due to N.
caninum (Jacobson and Jardine, 1993).
A combination of clindamycin (Antirobe, 10 mg kg-~ three times daily) and
trimethoprim plus sulfonamide (Trimabac 80, BK Products, United Kingdom, 15 mg
kg- ~ twice times daily) was used with success in a pup with paraplegia and pelvic limb
hyperextension (Knowler and Wheeler, 1995). The pup lived, but the hind limb
problems never resolved.
Clindamycin hydrochloride (Antirobe, UpJohn, Kalamazoo, MI, 7.5 mg kg-l for 45
days) therapy was successful in treating pyogranulomatous neosporosis dermatitis in a
12-year-old dog (Dubey et al., 1995). Numerous tachyzoites were present in dermal
ulcers before treatment, but live N. caninum was not found when the dog was
euthanized because of concurrent lymphosarcoma.
Barber and Trees (1996) reported data on the treatment of 16 dogs with neosporosis
with clindamycin, pyrimethamine, and sulfadiazine, either alone or in comination. Five
dogs made a full recovery. Good response to treatment was seen in five dogs.
Gastroenteritis was reported in 1 dog that received clindamycin, and two dogs that
received sulfadiazine and pyrimehamine.
Presently, there is no treatment that will prevent a bitch from transmitting N.
caninum to her offspring.
3.6. Experimental infections
Congenital neosporosis has been reproduced in dogs (Dubey and Lindsay, 1989b;
Cole et al., 1995b). However, classical signs of neosporosis, as observed in natural
cases, have not been reproduced experimentally in dogs. Culture-derived NC-1 isolate
tachyzoites were inoculated in pregnant bitches in two trials. In Trial 1, a laboratory
bred Beagle was inoculated SC and IM with 1.5 X 10 6 tachyzoites. The bitch remained
clinically normal and delivered eight fullterm pups, 28 days later. Pup 1 was born dead,
Pup 2 died 2 days later, and Pups 3 and 4 were euthanized at 2 and 3 days of age
because they were not nursing. Pup 5 was euthanized in a moribund condition on Day
20. Neospora caninum was isolated in cell cultures inoculated with tissues of each of
the five pups. Histologically, although all five pups had lesions, encephalitis and
myocarditis in association with tachyzoites was verified only in PUp 5 (Dubey and
Lindsay, 1989b). Pups 6 to 8 and the bitch remained clinically normal. They developed
hepatic lipidosis, pneumonia and myositis following administration of large doses of
corticosteroids (Dubey and Lindsay, 1990a). Neospora caninum tachyzoites were seen
in tissues of Pups 6 to 8 and the bitch, indicating that under certain circumstances
subclinical neosporosis can be reactivated.
In Trial 2, six random source bitches were inoculated SC with 5 106 tachyzoites at
21 day of pregnancy (Cole et al., 1995b). Four bitches had only macerated, mummified
or resorbed fetuses and it was not possible to demonstrate N. caninum in fetal tissues;
Fig. 6. Neospora caninum-induced lesions in fetal placenta from a bitch at 39 day of gestation. A. Focal
necrosis (arrow) in a fetal placental villus. H&E stain; Bar: 50 p.m.B. Similar lesion as in 6A but stained with
the ABC immunohistochemical stain. Bar: 50 p.m.C. Tachyzoites (arrowheads) present in lesion depicted in
6A. H&E stain; Bar: 10 p . m . D . Villus stained with the ABC immunohistochemical stain. Numerous
tachyzoites are readily visible (arrow) in groups. Bar:. 50 btm. E. Section of the same villus stained with H&E.
Note that few tachyzoites (arrow) are apparent in this preparation. Ba~. 50 p.m.F. Higher magnification of 6D
demonstrating numerous tachyzoites (arrowheads). Bar" 10 p.m.G. Higher magnification of 6E demonstrating
numerous tachyzoites (arrowheads). Ba~. 10 p.m.
one of these bitches died of disseminated neosporosis 17 DAI. One bitch gave birth to
three live fullterm pups, with mild neurologic deficits but N. caninum was not
demonstrated in tissues of pups killed 9-13 weeks after birth. The last bitch was killed
18 days after N. caninum inoculation because it had ultrasonic evidence of fetal death.
There were 12 fetuses or sites of fetal attachment. Three fetal sites were not investigated
because the fetuses were resorbed. Neospora caninum was found in tissues of the nine
remaining fetuses, two of which were alive at the time of necropsy. There were no
inflammatory lesions although numerous tachyzoites were seen in fetal tissues. Many
tachyzoites were degenerating along with fetal tissues. Sections of fetal placenta from
one of these fetal attachment sites are shown in Fig. 6. Necrosis and N. caninum
tachyzoites were found in maternal and fetal placenta.
These experimental studies indicate that N. caninum can cause early fetal death,
mummification, resorption, and birth of weak pups. We are not aware of any report of
abortion due to neosporosis in dogs.
In Trial 3, three of the eight 5-day-old pups inoculated SC with a total of 1 million
tachyzoites of the NC-1, NC-2 and NC-3 isolates, developed clinical neosporosis (Cole
et al., 1995b). One pup had myalgia, muscle atrophy of the pelvic limbs and persistent
cough and N. caninum was isolated from its brain and muscle tissue when the pup was
killed 27 DAI. Histologically, the pups had evidence of encephalitis, myositis, hepatitis
and interstitial pneumonia, and N. caninum tachyzoites were seen in lesions. Tissue
cystlike structures were seen in sections of brain. One pup was laterally recumbent and
was euthanized 37 DAI. Microscopic lesions were seen in the brain, heart, tongue,
skeletal muscles, lung and liver. A third pup died of neosporosis 12 DAI with
inflammatory and necrotic lesions and N. caninum tachyzoites in the brain, heart,
tongue, limb muscles, diaphragm, lung and liver. The results indicate that postnatally
infected pups can develop clinical neosporosis.
4. Neosporosis in cattle
4.1. History
Historically, Thilsted and Dubey (1989) first reported N. caninum-like organisms in

brain tissue of bovine fetuses from a herd with persistent abortions in New Mexico.
Initially, the provisional diagnosis was made based on finding a few small T. gondii-like
tissue cysts and the inability to find any other cause of abortion. Toxoplasmosis was
excluded because aborting cows had no T. gondii antibodies and T. gondii is not an
abortifacient for cattle. Finally, the diagnosis was confirmed when N. caninum specific
serum became available (Lindsay and Dubey, 1989c) and the parasite in bovine tissues
was found to react with N. caninum antibodies. Barr et al. (1990) were the first to
recognize T. gondii-like protozoa as a major cause of bovine abortion in California.
Using the anti-N, caninum specific serum (Lindsay and Dubey, 1989c), they found that
N. caninum is a major cause of bovine abortion in California drylot dairies (Anderson et
al., 1991, Barr et al., 1991a).
Retrospectively, the earliest recorded case of neosporosis in cattle appears to be in a

calf from New South Wales, Australia. The calf was fullterm and born dead in 1974
(Hartley and Bridge, 1975; Dubey et al., 1990a). The first case of neosporosis in a beef
calf was reported from Maryland by Dubey et al. (1990f). Neosporosis is now regarded
as a major cause of abortion in dairy cattle in several countries. Salient features of
neosporosis in cattle are as follows.
4.2. Prevalence of abortion
Neosporosis has been reported from Australia, Canada, Denmark, UK, Ireland, Israel,
Japan, Mexico, the Netherlands, New Zealand, South Africa, Sweden, the USA and
probably has a worldwide distribution. Individual or few cases of neosporosis-associated
abortion reported are summarized in Table 8.
Outbreaks of confirmed neosporosis abortion reported from Australia, England,
Ireland, Mexico, New Zealand, and the USA are summarized in Table 9.
In addition to the outbreaks listed above, serologic evidence was used to investigate
outbreaks of abortion. Trees et al. (1994) found high titer ( > 1:1280) IFAT antibodies in
sera of 11 of 120 cows obtained within 2 days after abortion in the UK. Such high titer
antibodies were not found in 97 non-aborted cows. On a farm in New Zealand, 33% of
Table 8
Summary of isolated reports of neosporosis associated abortions in cattle
Location Reference No. of Gestational Remarks
fetuses age (months)
Canada
British Columbia Mclntosh and Haines (1994) 1 8 Beef calf.
Prince Edward Island Bildfell et al. (1994) 1 6 Endemic abortion
in herd.
Denmark Agerholm and Barr (1994) 2 5, 7 Fetuses were from
herds with endemic
abortions.
Israel Harmelin et al. (1995) 3 NR 20 fetuses were
examined histologically.
Japan Ogino et al. (1992) 5 5, 9 4 fetuses were 5 - 7
months old and 1 calf
was 1 day old.
South Africa Jardine and Last (1993) 2 7 Twin fetuses were
affected.
Sweden Holmdahl et al. (1995) 1 4.5 Endemic neosporosis
in the herd.
USA
Illinois Shivaprasad et al. (1989) 1 5 Neospora was seen
in placenta. The cow
aborted in 1982 a.
Maryland Dubey et al. (19900 1 8 Massive myocarditis
in a beef calf.
a Earliest record of retrospective neosporosis abortion in cattle.

Table 9
Summary of epizootics of bovine abortion associated with neosporosis
Location Reference No. of Remarks
cases a
Australia
New South Boulton et al. (1995) 152 729 fetuses from 126 herds (59%
Wales dairy and 41% beef) from 1982-1994
were examined.
Tasmania Obendorf et al. (1995) 3 From 1 herd 5.4 to 8.2% of cows
aborted. Of 11 fetuses submitted, 8 had
lesions suspected of neosporosis.
Ireland McNamee and Jeffrey (1994) 2 11 of 150 Friesian Holstein dairy
cows from 1 herd aborted in 1993. Five
fetuses had inflammatory lesions
and N. caninum was
identified in 2.
McNamee et al. (1996) 14 21 of 335 fetuses had lesions
suggestive of neosporosis. Neo~pora-
infected fetuses were from 9 dairies.
Mexico Abbitt et al. (1993) 6 Fetuses were from a dairy of 800
Holsteins with persistent abortions.
Netherlands Wouda et al. (1992); 554 Of 2184 fetuses submitted to the
Wouda et al. (1995) Drachten Laboratory, the prevalence of
confirmed neosporosis was 18.6%,
15.8% and 18.0% of fetuses submitted
during 1992, 1993 and 1995, respectively.
New Zealand Thornton et al. ( 1991 ) 2 Lesions indicative of neosporosis
USA were seen in 28% of 320 aborted
fetuses submitted to one diagnostic lab.
New Mexico Thilsted and Dubey (1989) 2 29 of 240 cows from 1 herd aborted
in 5 months. Nine fetuses were
submitted, 7 had lesions typical
of neosporosis and N. caninurn
was demonstrated in 2 fetuses.
California Ban" et al. (1990); 66 80 of 445 fetuses from many dairies
Barr et al. (1991a) were examined.
California Anderson et al. (1991, 1992) 170 Of 698 fetuses submitted to 1
diagnostic laboratory, 24.4%
were confirmed neosporosis.
California Anderson et al. (1995) I 13 Fetuses were from 26 dairies
involving 19708 cows. In addition
to 42.5% confirmed neosporosis
abortion, an additional 6.4% of
fetuses had lesions suggestive
of neosporosis.
California McAIlister et al. (1996a) 7 N. caninum was identified in 7 of 8
fetuses from a dairy of 1400 cows.
Of 360 pregnant cows, 66 aborted
within 2 months. Evidence indicated
a point source exposure.
Table 9 (continued)
Colorado Rogers et al. (1993) 6 166 cows in a Holstein dairy herd
aborted in 3 years. Twenty fetuses
were submitted for diagnosis.
Midwestern Nietfeld et al. (1992) 19 655 fetuses were submitted from 15
states herds. One infected fetus was
from a beef herd.
South Dakota Yaeger et al. (1994) 7 9 of 90 cows from a herd of
Holsteins aborted during a 10 day
period. Two additional cows aborted
in the following 2 weeks. N. caninura
was identified in all 7 fetuses examined.
United Kingdom Dannatt et al. (1995) 1+ Neosporosis was diagnosed on a
dairy herd with persistent fetal and
stillbirth losses of > 10% over
5 years (see text).
Otter et al. (1995) 8 Of the 190 abortion cases from
several herds from England and Wales
20 (10.5%) had lesions suggestive
of neosporosis and 8 were confirmed
neosporosis.
a Fetuses with confirmed N. caninum infection.
the breeding stock of 158 cows aborted in the autumn of 1993 (Thornton et al., 1994).
Four more cows were found open in August. Neosporosis was confirmed in four aborted
fetuses from this farm submitted to the diagnostic laboratory. Neospora caninum
antibodies were found in 50% of the herd 5 months after abortion.
There is no doubt that immunohistochemistry (II-IC) is insensitive in diagnosing fetal
neosporosis and maternal serology might aid diagnosis. McNamee et al. (1996) com-
pared the IFAT titers in maternal sera from 40 cows whose fetuses had been examined
for neosporosis by IHC. Of the 22 IHC-confirmed cases, 21 cows had titers of ~ 1:640
whereas, of the 18 IHC negative cases, only one cow had a titer of > 1:640. Based on
these findings, they examined sera from 489 cows from several herds in Northern
Ireland and found that 12.6% of aborting cows and only 3% of control cows had IFAT
titers of > 1:640. Thus, they felt that 9% of cows (12% minus 3%) likely aborted due to
neosporosis. This is double the 4.2% abortion rate diagnosed by IHC.
4.3. Clinical signs
Abortion is the only clinical sign observed in infected cows. Cows of any age may
abort from 3 months of gestation to term. Fetuses may die in utero, be resorbed,
mummified, autolyzed, stillborn, born alive, but diseased, or born clinically normal but
chronically infected.
4.4. Season and abortion
Neosporosis-induced abortions occur year round (Anderson et al., 1991; Tllurmond et

al., 1995b; Moen and Wouda, 1995). In California, more cases were found in winter
than in summer and early fall (Anderson et al., 1991; Thurmond et al., 1995b). In the
Netherlands, the N. caninum-induced abortion rate was higher in late summer and early
fall (Moen and Wouda, 1995).
4.5. Abortion in dairy vs. beef cows
Both dairy and beef cattle are affected although most reports are from dairy cattle.
Most reports of abortion from California were from drylot dairies (Barr et al., 1991a;
Anderson et al., 1991, Anderson et al., 1995), while most in the Netherlands were from
pastured dairy cattle (Wouda et al., 1995). In one study from Australia that examined
729 aborted fetuses from 126 affected herds, 59% of the affected herds were dairy and
41% were beef herds (Boulton et al., 1995). Neosporosis abortions has been induced in
beef cows (Ban" et al., 1994b).
4.6. Age of the aborting cow
Abortion due to neosporosis have been reported in animals up to 8 year old. Whether
there is an age-related susceptibility to neosporosis is unknown. In one epizootie in New
Zealand where 33% of the breeding herd aborted in autumn of 1993, abortions were
most common in cows up to 4 years old (Thornton et al., 1994).
4.7. Age of the fetus
Fetal death probably occurs throughout the gestation period, although abortion of
fetuses younger than 3 months of age have not been reported. It is likely that 1 to
2-month-old fetuses are killed in utero, resorbed, and the cow returns to heat again. The
mean gestational age of N. caninum abortion in California was 5.5 months (Anderson et
al., 1991). In one study, of 113 confirmed Neospora abortions, three fetuses were 3
months old; 27 were 4 months old; 33 were 5 months old; 22 were 6 months old; 16
were 7 months old; eight were 8 months old; and four were of 9 months gestational age
(Anderson et al., 1995). In the Netherlands study, the interval between insemination and
abortion was 110 to 259 days with peak gestational abortion age being 170 days (Moen
and Wouda, 1995).
4.8. Epizootic or sporadic abortion
Cows may abort sporadically or in groups within a few weeks or abortions may
persist in a herd. There are several reports of abortion storms (Thornton et al., 1994;
Yaeger et al., 1994; Anderson et al., 1994; Moen et al., 1995a; McAllister et al., 1996a).
In one outbreak, 33% of the breeding herd aborted within a few months (Thornton et al.,
1994). In most outbreaks, diagnosis was based on histologic examination of only a few
fetuses. Therefore, it is uncertain if all abortions in a given outbreak were due to
neosporosis.
Only rarely, have all aborted fetuses from participating dairies been examined for
abortifacients. In a study of 26 drylot dairy herds in California, involving 19708 cows,
N. caninum was identified in 113 (42.5%) of 266 cases of abortions over a period of 1
year. In addition to 42.5% confirmed neosporosis abortion of this study, there were 6.4%
suspected cases of neosporosis abortions based on lesions and exclusion of other causes.
Bacterial and mycotic infections accounted for 14% of abortions and 37.2% of abortions
were not diagnosed (Anderson et al., 1995). In another episode, 18% of cows aborted
during a 6-week period and all 17 fetuses examined were diagnosed to have N. caninura
(Anderson et al., 1994).
Moen et al. (1995a) reported explosive outbreaks of neosporosis abortion in four
dairy herds of Holstein pastured cattle from the Netherlands. These herds had 227
milking cows and 99 heifers that were at risk of abortion. The abortion storms lasted
approximately 3 weeks and were followed by the birth of mummified fetuses in
subsequent months. The abortion rate was higher in cows (33% of 227) than in heifers
(14% of 99). Of 51 fetuses submitted for diagnosis, lesions suggested of neosporosis
were found in 50 and the diagnosis was confirmed by IHC in 40. The fertility following
abortion was not affected. Of subsequent pregnancies followed in 71 cows, all but six
cows had clinically normal calves. There were three stillborn calves, but the cause of
stillbirth was not determined. Three cows aborted a confirmed Neospora-infected fetus
the second time, one in the first, one in the second and one in the third pregnancy
following abortion. Thus, < 5% of cows aborted a Neospora-infected fetus for the
second time. Precolostral antibodies were found in 68% of the clinically normal calves
tested indicating a high rate of congenital transmission of N. caninum. Seronegative
cows produced seronegative calves.
4.9. Repeat abortions
Neospora caninum can cause repeated abortions in the same cow (Obendorf et al.,
1995; Wouda et al., 1995; Anderson et al., 1995; Dannatt et al., 1995; Moen et al.,
1995a). However, the frequency of repeat abortions due to confirmed neosporosis is
unknown because many aborted cows are culled (Obendorf et al., 1995). One cow
aborted in each of three successive pregnancies in 1987, 1988, 1989; N. caninum was
identified in fetal tissues from the 1987 and 1989 pregnancies (Obendorf et al., 1995).
The other known instance of confirmed repeated abortion was reported by Anderson et
al. (1995). Of 112 cows with confirmed N. caninum-induced abortions, four had twice
aborted N. caninum infected fetuses. Thus, < 4% had aborted twice due to neosporosis.
In the Netherlands study, two of 26 cows aborted confirmed N. caninum infected
fetuses for the second time (Wouda et al., 1995). In a New Zealand study, there were
only two repeat abortions (cause not investigated) in a herd o f 158 cattle that had
experienced a major (33%) episode of presumed N. caninura abortion (Thornton et al.,
1994). As said earlier < 5% of cows aborted Neospora-infected fetus for the second
time (Moen et al., 1995a).
In a dairy herd from Northern Ireland, 31 cows aborted during the 1993-1994
calving season. Five cases were confirmed as being caused by N. caninum. In following
calving season only three cows from this herd aborted (McNamee et al., 1996).
In a 95 cow dairy herd in Cornwall, England, ten cows aborted within a period of 39
days (Dannatt et al., 1995). Nine of the ten aborting cows had IFAT titers of > 1:640
and diagnosis was confirmed by IHC on one fetus. Two weeks after the last abortion,
60% of the 95 cows had IFAT titers of > 1:640. One of the nine cows that aborted and
had an IFAT titer of 1:5120 aborted again. This fetus was IHC positive for N. caninum;
unfortunately the first fetus was not examined. In the following year, six of the nine
cows produced clinically normal calves and three were culled from the herd. Thus, one
cow had evidence of repeat abortion due to N. caninum.
4.10. Fetal lesions and pathogenesis of abortion
Degenerative to inflammatory lesions may be found throughout fetal tissues, but are
most common in the CNS, heart, skeletal muscle, and liver (Barr et al., 1990, Barr et al.,
1991a; Anderson et al., 1991; Wouda et al., 1996). Gross lesions are rare, but may be
present in heart, skeletal muscles and the brain. In a fullterm stillborn calf, the heart was
enlarged (Dubey et al., 1990f). Pale white foci may be present in skeletal muscles and
the heart. Minute pale to dark foci of necrosis in the brain may occur. Often the fetuses
are autolyzed and mummified. Neural lesions consist of nonsuppurative encephalomyeli-
tis characterized by multifocal nonsuppurative infiltration, with or without multifocal
necrosis and multifocal to diffuse nonsuppurative leukocytic infiltration of the meninges.
The characteristic lesion of neosporosis in the CNS consists of a focus of mononuclear
cell infiltration around a central area of necrosis (Fig. 7A). Glial proliferation is more
common in fetuses aborted in the 3rd trimester. In one study of 82 fetuses, encephalitis
and myocarditis were seen in 100%, adrenalitis in 80%, myositis in 72%, nephritis in
66%, hepatitis in 62%, placentitis in 53% and pneumonia in 44% (Barr et al., 1990).
Although N. caninum was not identified in all of these fetuses at that time the character
and distribution of lesions due to neosporosis have not changed. In a recent study of 80
aborted bovine fetuses from the Netherlands with parasitologicaily confirmed neosporo-
sis, histologic lesions and the distribution of parasites in the brain, heart and liver were
compared. In 83 cases (91%), lesions were present in each of the three organs, whereas
in the remaining seven cases, lesions were seen in two of the three organs. Brain lesions
were seen in all but one case and consisted of nonsuppurative encephalitis with
multifocal necrosis in 76% of cases. Occasionally, there was calcification which was
also observed by Boulton et al. (1995).
Myocardial lesions are severe, but are often masked by autolysis. In two well-pre-
served fetuses, there was extensive myocarditis (Fig. 8) and necrosis of cardiomyocytes
(Dubey et al., 1990f; Wouda et al., 1996).
Hepatic lesions consist of periportal infiltrations of mononuclear cells and variable
foci of hepatocellular necrosis (Barr et ai., 1990; Wouda et al., 1996).
Neospora caninum is most often demonstrable in the brain and heart and rarely in
other organs, including the placenta. In the study by Wouda et al. (1996), N. caninum
was found in the brain of 71 (89%), heart of 11 (14%) and in the liver of 21 (26%) of 80
fetuses examined by IHC. Wouda et al. (1996) also found that there were more N.
caninum organisms in epizootic versus sporadic cases of abortion.
Although little is known of the tissue cyst formation in the bovine fetus, in one
experimentally infected fetus a small (10/zm) tissue cyst was found in brain of the fetus
whose dam had been inoculated with N. caninum 32 days before euthanasia of the fetus
Fig. 7. Lesions in CNS of cattle naturally infected with N. caninum. A. Fetal brain with an inflammatory
lesion with a central necrotic focus. H&E stain. Bar: 50/zm. B. Spinal cord of a congenitally infected calf.
Note glial proliferation and perivascular inflammation and two tissue cysts (arrows) at the periphery of the
lesion. Immunohistochemical stain with anti-N, caninum antibodies; Bar: 50 p,m.
( D u b e y et al., 1992c). T i s s u e cysts w e r e not reported in b o v i n e fetal tissues r e m o v e d

a p p r o x i m a t e l y 1 m o n t h after inoculation of the c o w s with N. c a n i n u m b y Barr et al.
(1994b).
34 J.P. Dubey, D.S. Lindsay/ VeterinaryParasitology 67 (1996) 1-59
Fig. 8. Lesions in the myocardium of a naturally infected bovine stillborn calf. A. Inflammatory lesion with
several large (arrows) groups of tachyzoites. Immunohistochemical stain. Bar: 50/.tin. B. Necrotic focus with
a large group of tachyzoites (arrow). Note the absence of a cyst wall. H&E stain; Bar: l0 /xm.
4.11. Diagnosis of bovine neosporosis fetal abortion

Examination of maternal and fetal sera, a n d o f fetal tissues, can aid in diagnosis. T o
the present, diagnosis has been based mainly on histologic e x a m i n a t i o n o f fetal tissues
stained with N. caninum specific antibodies. Ideally, maternal sera and the w h o l e fetus
should be submitted for diagnosis. If there is a problem in submitting the whole fetus,
the head can be submitted to the diagnostic laboratory. At present, there are no data on
preferential localization sites of N. caninum in bovine brain. Any part of the brain may
be used for histologic examination. Even in severely autolyzed fetuses, some solid
portion may be found. Neospora caninum has been found in mummified fetuses and in
fetuses where the brain tissue is almost liquid. Neospora caninum stages are few and
difficult to find in ordinary hematoxylin and eosin stained sections~ Staining with N.
caninum specific antibodies helps to locate organisms. As mentioned earlier, N.
caninum tachyzoites in fetal brain are often few in number and are often dead.
Therefore, it is difficult to locate well-preserved tachyzoites (Nietfeld et al., 1992).
Because some host cells might be stained with N. caninum antibodies nonspecifically, it
is important to locate structures with typical morphology of tachyzoites (Fig. 2). Tissue
cysts in fetal brains may be small in size and may not have developedthe characteristic
thick cyst wall of N. caninum (Fig. 2). Differential diagnosis includes infection by T.
gondii and S. cruzi. There is as yet no documented case of naturally occurring
toxoplasmosis abortion in cattle. Therefore cross reactivity with T. gondii is not a
practical problem with reference to bovine neosporosis. Sarcocystis-induced abortion in
cattle is rare (Anderson et al., 1991). Unlike N. caninum, S. cruzi schizonts are found
primarily in the vascular endothelium. Ultrastructurally, merozoites in Sarcocystis
schizonts lack rhoptries (Dubey, 1993a). Immunohistochemically, N. caninum antibod-
ies do not stain S. cruzi schizonts.
Neospora caninura from the aborted material may be isolated in cell cultures.
However, the success rate is very low because of autolysis and most N. caninum
organisms probably die along with host tissue. Neospora caninum was isolated from
only two of 100 bovine fetuses with histologically confirmed neospomsis (Conrad et al.,
1993a). In both instances, tissue cysts were present in histologic sections made from the
donor fetal brains. It is probably easier to isolate N. caninum from neural tissues of
congenitally infected fullterm calves because more tissue cysts are likely to be present
and tissue cysts are more resistant than tachyzoites. Neospora caninum grows in many
cell lines, therefore, the choice of cell line to isolate the parasite is probably not critical.
However it is important to observe cell cultures for about 2 months because only a few
parasites may be present in the inoculum and it may take a while for the parasite to
adapt to cell culture.
On two occasions N. caninum was isolated in the brains of mice inoculated with
aborted fetal brain (Obendorf et al., 1995) and from a congenitally infected calf (Bryan
et al., 1994). Use of inbred mice (BALB/c) rather than outbred (Swiss-Webster) may
facilitate isolation of N. caninum from bovine fetuses, especially where the material is
unsuitable for cell culture because of microbial contamination.
Finding N. caninum specific antibodies in ruminant fetuses is useful in diagnosis,
especially in autolyzed fetuses. The chance of finding N. caninum antibodies in fetuses
are increased with gestational age. Barr et al. (1995) found N. caninum antibodies at a
1:80 dilution in the IFAT in 37 of 74 fetuses with suspected or confirmed neosporosis-
associated abortion; 31 of these fetuses with N. caninum antibodies were 6 months or
older in gestational age. Neospora caninum antibodies were detected in only one of 64
fetuses which aborted due to other causes, suggesting the usefulness and specificity of
36 J.P. Dubey, D.S. Lindsay / Veterirvary Parasitology 67 (1996) 1-59
the test. Antibodies to T. gondii were not detected in any of the 135 fetuses, with or
without N. caninum antibodies. In their study, Barr et al. (1995) found no correlation
between the presence of N. caninum antibodies and the increase in total IgG ( > 15 mg
dl -~) that is often associated with microbial infections. Detection of N. caninum
antibodies in aborted fetuses is not likely to be due to 'passive' passage of maternal
antibodies from placenta damaged by other microbes. In experimentally infected ewes
non-T, gondii antibodies were not transferred through a ruminant placenta damaged by
T. gondii (Dubey et ai., 1987). It must be emphasized that failure to find N. caninum
antibodies does not rule out neosporosis because the fetus may not have been immuno-
competent (-<4 months) and there may not have been enough time between fetal
infection and abortion. The type of test used and the screening dilutions are also
important. Barr et al. (1995) screened sera at a 1:80 dilution in an IFAT. Thus the
threshold titer indicative of N. caninum infection in fetuses has not been determined.
Blanchard et al. (1995) questioned the usefulness of fetal serology in the diagnosis of
bovine abortions because they found N. caninum antibodies in 17% of fetuses with
confirmed neosporosis and in 18.6% of fetuses suspected to have been aborted due to
causes other than N. caninum. Blanchard et al. (1995) screened sera in an ELISA with a
cut off optical density (O.D.) value of 0.25 which was set lower than the 0.45 O.D.
value used for adult cattle sera (Par~ et al., 1995b). Reichel and Drake (1996) found
good correlation between IFAT (titer > 1:200) and ELISA (O.D. > 0.45) in bovine sera
and fetal fluids but poor correlation with histologic diagnosis of Neospora abortion in
New Zealand.
Buxton et al. (1995) examined 451 fetal bovine sera for N. caninum IgG antibody
and found 12% of aborted fetuses to have IFAT titers of 1:64 to 1:16384. One hundred
and sixteen of the sera were also examined for IgM and IgG antibody and 15 (12.9%)
were positive by IgG-IFAT, 11 (9.5%) were positive by IgM-IFAT, and six were
positive with both tests.
4.12. Neosporosis in congenitally infected calves
Neosporosis-infected calves may have neurologic signs, be underweight, unable to

rise or be born without clinical signs. Hind limbs a n d / o r forelimbs may be flexed or
hyperextended. Neurologic examination may reveal ataxia, decreased patellar reflexes,
and loss of conscious proprioception (Parish et al., 1987; Barr et al., 1993). Although
subclinical congenital neosporosis is probably common, only a few cases with clinical
neosporosis have been reported (Table 10). Calves may have exophthalmia or asymmet-
rical appearance of eyes (O'Toole and Jeffrey, 1987; Bryan et al., 1994), or deformities
associated with insult of embryonic neural cells (Dubey and de Lahunta, 1993).
In all congenitally infected calves born live, the disease was confined mainly to the
CNS. Gross lesions consisted of malacia (O'Toole and Jeffrey, 1987), and deviation or
narrowing of the vertebral column (Dubey et ai., 1990a; Barr et al., 1991b; Bryan et al.,
1994). Microscopic lesions consist of nonsuppurative encephalomyelitis characterized by
perivascular cuffs, gliosis and mild necrosis (Fig. 7B). Tissue cysts are frequently
observed (Table 10). The most tissue cysts seen were in the 3-day-old calf reported by
Barr et al. (1991b). Generally, tissue cysts are not present in every section of the lesion
Table 10 g:
Summary of calves clinically affected with congenital neosporosis
Location Reference No. of Day old when Clinical Lesions in Tissue Tachyzoites Breed
calves necropsied signs CNS b cysts
Canada
Alberta Bryan et al. (1994) 1 3 Moribund, Yes, congenital Yes No Salers
limbs flexed deformity "x
United Kingdom
Gioucestershire, O'Toole and Jeffrey (1987); 1 5 Yes Yes No Yes Friesian
England Dubey (1989)
USA
Washington State Parish et al. (1987); 4 3 Paralysis Yes Yes Yes 3 Herefords,
Dubey et al. (1989) I Angus-Simmental
California Barr et al. (1993) a 3 2-6 Ataxia Yes Yes No Holstein
Barr et al. (1991b) 1 3 Ataxia Yes Yes Yes Holstein
New York Dubey and de Lahunta (1993) 1 14 Yes Yes Yes Yes Holstein
a Calves had precolostral N. caninum antibodies.

b Lesions were not seen in extra neural organs.
38 J.P. Dubey,D.S. Lindsay/ Veterinary Parasitology 67 (1996) 1-59
and there appear to be more N. caninum in the spinal cord than in the brain. In all
congenitally infected calves N. caninum was confined to the brain and spinal cord.
Myositis and limb deformities were considered to have resulted from muscle degenera-
tion (Dubey and de Lahunta, 1993).
In addition to the calves reported in Table 10, Barr et al. (1993) diagnosed
neosporosis in two Holstein calves, based on the presence of high precolostral N.
caninum antibodies. One calf was 2 days old and the other was 17 days old. Neospora
caninum was not found in tissue sections of any of these two calves, but the 17-day-old
calf had a mild nonsuppurative encephalomyelitis.
Dubey et al. (1992b) reported neosporosis in a 4-week-old Hereford calf. The calf
was fullterm and appeared clinically normal at birth. At 2 weeks of age, the calf
developed neurologic signs. The report on this calf is unusual because this is the oldest
reported calf with clinically diagnosed neosporosis. It had extensive neural lesions with
numerous associated N. caninum tachyzoites, and lesions in skeletal muscles also
associated with tachyzoites of the parasite. Whether the calf was prenatally or postna-
tally infected is unknown. It is likely that most calves with clinical neosporosis die
within the first 4 weeks of life. This situation parallels congenital ovine toxoplasmosis
where lambs may be aborted, stillborn, underweight, and die within 2 weeks of birth
(Dubey and Beattie, 1988). Many congenitally infected lambs are born clinically normal
and remain subclinical through life. Whether the development of clinical neosporosis in
calves is related to the strain of N. caninum, the gestational age of the fetus, or the
immune status of the dam at the time of infection is unknown.
Recently, Yamane et al. (1996) in Japan isolated N. caninum in cell culture
inoculated with neural tissue from a 2 week old clinically normal calf.
4.13. Repeat congenital infection
Repeat congenital neosporosis is probably more common than repeat Neospora

abortion. Five congenitally infected calves were born from 4 cows that previously had
Neospora infected fetus (Barr et al., 1993); three of these five calves had Neospora in
their brain and the other two were diagnosed on the basis of high N. caninum antibody
titers in precolostral serum. It is likely that only a small percentage of congenitally
infected calves have clinical neosporosis. On two farms in California, 31 and 54% of
calves had precolostral N. caninurn antibodies (Par6 et al., 1996a).
4.14. Detection of N. caninum antibodies in cows
Detection of N. caninum-specific antibodies in the sera of cows can be useful in the

diagnosis of bovine abortion and to study the seroepidemiology of neosporosis and both
the IFAT and ELISA have been used. In the IFAT for neosporosis, cell culture derived
whole tachyzoites are used and the antigen slides are commercially available (VMRD,
Pullman, WA, USA). There are three important observations when considering the
specificity of the N. caninum IFAT: The serum in the medium used to grow cells for N.
caninum tachyzoites, the pattern of fluorescence, and the screening dilution. Normally,
fetal calf serum is used in the cell culture medium. Because of the high prevalence of
subclinical fetal neosporosis, most batches of commercial fetal calf sera have N.
caninum antibodies, which will interfere with the IFAT. Furthermore, binding of
non-specific immunoglobulins or other proteins in culture media to tachyzoites can be
recognized non-specifically by anti-bovine conjugates. The calf serum can be replaced
by goat or horse serum free of N. caninum antibodies, or serum can be omitted towards
the end stage of culture. Although the pattern of fluorescence is subjective, it is
important that for a positive test the whole tachyzoite surface should fluoresce because
nonspecific antibodies can produce partial surface (apical) fluorescence (Conrad et al.,
1993b; Par~ et al., 1995a). The spectrum of antibodies that produce this cross reaction
has not been determined. Agents to which cattle are exposed after birth probably are
involved in this cross reactivity because the apical fluorescence was not observed with
fetal bovine sera (Barr et al., 1995).
The screening dilution is also important. For fetal sera a 1:80 dilution was considered
specific, although sera were not tested at lower dilutions (Barr et ai., 1995). For adult
cattle a dilution in the range of 1:200 appears to be N. caninum specific. Of 61 adult
cattle with no history of N. caninum infection, 53 (87%) had titers of < 1:160, seven
had titers of 1:160, and one had a titer of 1:320 (Conrad et al., 1993b). Of 64 cows that
aborted N. caninum infected fetuses, 63 had titers of >- 1:640 and one had an IFAT of
1:320. Conversely, of nine cows with no history of N. caninum abortion, eight had titers
of < 1:160 and one had a titer of 1:320. Thus, an IFAT titer of 1:640 was considered
specific for N. caninum by these authors (Conrad et al., 1993b). In order to investigate
the feasibility of diagnosing neosporosis in clinically normal but congenitally infected
cattle by measuring antibodies in precolostral serum, Par6 et al. (1995a) compared IFAT
titers (cut off titer 1:640) in 189 pairs of calves and their dams. From 62 seropositive
cows there were 52 seropositive calves. From 127 seronegative cows there were 16
seropositive calves. Because antibodies of the cow may be sequestered in the colostrum
at the time of parturition, it is possible that periparturient antibody titers of cows were
temporarily reduced. Furthermore, the cut-off IFAT titer ( ~ 1:640) may have been too
high, thus resulting in false seronegative results in 16 cows that gave birth to seronega-
five calves. Nevertheless, studies bY Par~ et al. (1995a) indicated that clinically normal
cattle can have very high IFAT titers. Of the 541 sera from two dairy and two beef herds
tested, 6% of adult cattle had titers of ~- 1:5120 (Par6 et al., 1995a).
Only limited information is available concerning the persistence of IFAT titers in
naturally exposed cattle. Of six cows (with titers of > 1:640) with a confirmed N.
caninum-infected aborted fetus, 1FAT titers had declined to 1:160 within 5 months
(Conrad et al., 1993b). The titer of one cow rose from 1:160 to 1:1280 when rebred, this
cow gave birth to a congenitally infected calf. These observations indicate that titers can
fluctuate during pregnancy; whether it was due to reactivation or reinfection could not
be determined.
Experimentally-infected cattle can develop IFAT titers to N. caninum within 4 weeks
of tachyzoite inoculation (Conrad et al., 1993b; Barr et al., 1994b; Dubey et al., 1996a).
In a study from California, six pregnant heifers seroconverted from < 1:80 to 1:640
within 7 to 22 days of inoculation with a bovine isolate of N. caninum (Table 11).
Similar results were obtained in six cows inoculated with canine isolates of N. caninum
Table 11
Antibody titers (IFAT) in cattle inoculated with a bovine isolate of N. caninum a
Heifer Seropositivity Fetus
No. Day seropositive Day peaked Peak titer Day after dam inoculation Titer
2A 14 30 1280 30 160
2B 14 30 640 ND ND
3A 22 39 640 ND ND
3B 11 46 10240 67 1280
4A 7 32 5120 31 640
4B 7 32 10240 155 20480
a Data from Barr et al. (1994b).

ND: No data.
(Dubey et al., 1996a). Data from two cows observed for two pregnancy cycles are
shown in Table 12.
Par~ et al. (1995b) used a crude lysate of N. caninum tachyzoites as antigen in the
ELISA to detect antibodies in bovine sera. At a serum dilution of 1:200 and 200 ng per
well N. caninum protein lysate and a cut off O.D. value of 0.45, they found good
correlation with sera of cattle that had IFAT titers of 1:1280. They also found that the
isolate of N. caninum, whether from dogs (NC-1) or from cattle (BPA-I), did not affect
the outcome of results.
In order to examine reactivity between N. caninum and related apicomplexans in
bovine sera, serial serum samples were obtained from six pregnant cows and two calves
inoculated parenterally with N. caninum, 12 calves inoculated orally with sporocysts of
Sarcocystis (eight with S. cruzi, two with S. hirsuta, two with S. hominis), six pregnant
Table 12
Neo~pora caninum antibodies in two cows inoculated with N. caninum a
PID Cow 2 Cow 3
IFAT ELISA (O,D.) IFAT ELISA (O.D.)
0 < 50 b ND 50 ND
22 1600 ND 3200 1.136
33 1600 ND 3200 ND
64 1600 1.265 6400 1. 100
85 1600 1.211 3200 0.765
92 1600 1.156 3200 0.761
123 400 0,860 1600 0.551
274 200 0.543 ND ND
375 ND 0,423 800 ND
449 800 0.480 ND ND
459 200 0.402 ND ND
516 400 ND ND 0.467
a Toxoplasma gondii modified agglutination test titers were < 50 for all cow sera.
b Reciprocal titers.
ND: Not done; PID: Postinoculation day. From Dubey et al. (1996a).
cows and eight calves inoculated orally with T. gondii oocysts, one calf inoculated with
Eimeria boris, and 12 calves inoculated with Cryptosporidium parvum (Dubey et al.,
1996a). In addition, sera from nine cows with histologically confirmed N. caninum-in-
fected fetuses were also examined. The screening dilution for IFAT for both N. caninum
and T. gondii was 1:50. For the ELISA, a lysate of N. caninum tachyzoites and a
screening serum dilution of 1:50 were used. Sera were also examined for T. gondii
antibodies by the MAT. All six cows and two calves with no preinoculation antibodies
developed N. caninum antibodies both in IFAT and ELISA within 4 weeks postinocula-
tion. Antibody titers of two cows are shown in Table 12. There was no cross reactivity
between T. gondii (using both the IFAT and MAT) and N. caninum in sera of cows
naturally or experimentally infected with N. caninum. Results were similar with sera
from calves experimentally infected with N. caninum, except for three sera that reacted
to T. gondii antigen (two sera at a l:100 dilution in the IFAT, and a 1:50 dilution in the
MAT; one serum at a 1:100 dilution in the IFAT but not at a 1:25 dilution in the MAT).
There was no cross reactivity between S. cruzi and N. caninum in the IFAT but there
was reactivity by the ELISA. Sera from cattle infected with T. gondii, Cryptosporidium
parvum and Eimeria boris did not react with N. caninum antigen in the ELISA or IFAT
(Dubey et al., 1996a).
Trees et al. (1994) did not find antiW, caninum IFAT titers at 1:640 in cattle
experimentally infected with T. gondii, S. cruzi, Babesia divergens, Eimeria boris, E.
albamensis, and Cryptosporidium parvum. In 20 sera from naturally infected cattle with
IFAT titers of > 1:1280, anti-T, gondii antibodies were not found in 19 sera and one
had a titer of 1:80 in the MAT.
Because nearly 100% of cattle are exposed to Sarcocystis in the USA and elsewhere,
there is a need to improve the specificity of the N. caninum ELISA. Some progress is
being made in this direction. Fusion proteins from two N. caninum recombinants
(Nc4.1, Ncl4.1) identified by immunoscreening with N. caninum positive cattle sera
appear promising for diagnosis (Lally et al., 1996a). The Nc4.1 clone encodes a
recombinant 35 KDa protein and the Nc14.1 clone encodes a 30 KDa recombinant
protein. Both fusion proteins reacted with sera from cows both from natural and
experimental infections (Lally et al., 1996a). Sera collected from cows by 103 DAI did
not react with these fusion proteins. Thus, these antigens may be useful in the detection
of acute neosporosis.
By using both ELISA and immunoblotting techniques, information may be obtained
relating to anti-N, caninum titers or a particular binding pattern to disease status.
Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with
confirmed neosporosis abortion showed at least 14 major antigens ranging in molecular
weight from l l to 75 KDa (Baszler et al., 1996). Antigens of 116, 65 and 25 KDa were
detected in all N. caninum aborted cows. Baszler et al. (1996) developed and character-
ized an N. caninum tachyzoite monoclonal antibody (MAb-4A 4-2) that bound to the
exterior surface of N. caninum tachyzoites and recognized the 65 kDA protein. A
competitive inhibition ELISA using this monoclonal antibody appears to be promising
for the diagnosis of neosporosis-induced abortion in cattle (Baszler et al., 1996).
Williams et al. (1996) developed an ELISA using whole formalin-fixed N. caninum
tachyzoites as antigen and a monoclonal antibody to bovine immunoglobulin light chain
with an anti-mouse horseradish peroxidase conjugate to reveal the bound antibody. At an

O.D. value of 0.77 there was a good agreement between ELISA and IFAT. No cross
reaction was observed using sera from cattle experimentally infected with a number of
apicomplexans.
BjiSrkman et al. (1996a) described an iscom-ELISA to detect antibodies to N.
caninum in sera and milk samples of cattle.
4.15. Seroepidemiology of bovine neosporosis
Control of bovine neosporosis may not be possible until the full life cycle of N.
caninum is known and sources for postnatal infection are identified. In the meantime,
information gathered by seroepidemiology may be useful for developing strategies to
control neosporosis.
At present, congenital transmission is the only known natural mode of N. caninum
transmission in cattle. The prevalence of N. caninum infection in some dairy and beef
herds is high (Par~ et al., 1994, Par~ et al., 1995a; Thurmond and Hietala, 1995). In a
report from Sweden, N. caninum antibodies (IFAT titers __. 1:640, ELISA O.D. > 0.25)
were found in 16 of 50 cows from a closed dairy herd (Bj/Srkman et al., 1996b). All the
seropositive animals in this herd were found to be progeny of two cows brought to the
farm in 1980. Two seropositive cows had two seropositive heifers born in two
successive pregnancies. A significantly higher mortality was found in progeny of the
seropositive cows than from the seronegative cows.
In seroepidemiologic studies from California, congenital infection was found in 78%
to 88% of calves born to seropositive cows in some dairies (Par6 et al., 1996a). Risk of
abortion for infected cows has been estimated to be twice that of noninfected cows
suggesting that half of the abortions in infected cows may be in theory attributable to
neosporosis (Par6 et al., 1996b). In a seroepidemiologic survey of four herds with
epidemic (Herds 1 and 2) or endemic (Herds 3 and 4) abortion considered due to
neosporosis, seropositivity was 20 of 34 (58.8%) for cows that aborted and eight of 45
(17.7%) for cows that did not abort in Herds 1 and 2. For Herds 3 and 4, ten of the 12
(83.3%) cows with a confirmed N. caninum abortion had seropositive heifers whereas
only one of 28 (3.6%) seronegative cows had a seropositive heifer. The authors
suggested that cows aborting a fetus infected with N. caninum were likely to have been
infected congenitally, and that abortion was not a consequence of recent exposure from
the environment (Thurmond et ai., 1995a). These authors also found a very low (1.1%
and 2.3%) incidence of postnatal N. caninum infection in two California herds. Until
more is known of the interpretation of ELISA, N. caninum titers in naturally-infected
cattle, and of the life cycle of the parasite, control measures should not be planned based
on these seroepidemiologic studies.
Long term seroepidemiologic studies in California have indicated that congenitally
infected calves born clinically normal did not experience a higher mortality compared
with seronegative calves (Par6 et al., 1996a). These studies were conducted on two
dairies (A and B) that experienced a high abortion rate due to neosporosis. Precolostral
samples were collected from calves and matched with samples from their dams. Later,
antibodies to N. caninum were assessed in the ELISA at a cut off O.D. value of 0.45
using a 1:200 dilution of serum (Par6 et al., 1996a). On Dairy A, 85 of 278 (30.6%)
calves and 82 of 228 (36.0%) cows had N. caninum antibodies and eight seropositive
calves were born from seronegative cows. On Dairy B, 68 of 127 (53.5%) calves and 33
of 57 (57.9%) cows had N. caninum antibodies and all seropositive calves were born to
seropositive cows. The probability of a calf being congenitally infected was not
associated with dam age, dam lactation number, dam history of abortion or calf gender
or length of gestation (Par6 et al., 1996a).
In a study from the Netherlands, of the 150 postabortion sera from confirmed N.
caninum abortion cases, 61% had high antibody titers (1:400-1:6400), whereas 21% had
low titers (1:50-1:200) and 18% had no detectable antibodies in their ELISA (Wouda et
al., 1995). Animals aborting during a major outbreak had a relatively low antibody
response. Neospora caninum antibodies were found in only one of the 50 cows aborting
from other causes. It is worth noting that unlike the seroepidemiologic studies of
Thurmond et al. (1995a) from California, the results from the Dutch study are based on
histologically confirmed cases of neosporosis.
Forty-two animals that aborted during a major outbreak in Netherlands were rebred.
Antibody titers declined to low or undetectable levels within 2 months after abortion.
Antibody titers were elevated in 62% (26 cows) of the cows during the second half of
gestation, indicating reinfection or reactivation. Two cows aborted a N. caninum-in-
fected fetus for the second time. Twenty-two cows gave birth to clinically normal calves
with precolostral N. caninum titers, indicating congenital infection (Wouda et al., 1995).
The observations by McAllister et al. (1996a) in a California dairy are important. A
large dairy herd with 1400 cattle experienced an acute outbreak of abortion in the fall of
1992. Because of the large size of the herd it was not possible to record all abortions and
obtain blood from all cows. Abortions in a sample of 360 pregnant cows were recorded
during the first 60 days of the outbreak. Sixty-six (18%) of these cows aborted.
Mummified fetuses were first observed on day 44 of the outbreak. A total of eight
aborted fetuses were examined during the first 72 days of the outbreak and all had
histologic evidence of neosporosis. Seventy-four cows from one pen were selected for a
seroepidemiologic study. Neospora caninum IFAT antibodies ( > 1:320) were found in
65 of 74 cows bled on Day 18 of the outbreak. Antibody titers of the aborted (cows)
were: 1:320 (one), 1:640 (two), 1:1280 (nine), 1:2560 (six), 1:5120 (four), 1:10240
(three) and of nonaborted cows were 1:80 (two), 1:160 (seven), 1:320 (six), 1:640 (13),
1:1280 (17), 1:2560 (three) and 1:5120 (one). Of the four cows bled four times (Days 4,
18, 39 and 65) during the abortion episode, Neospora antibody titers had peaked to
1:1280 or 1:2560 in three cows on Day 4 and 1:2560 in one cow on Day 18 of the
outbreak. The IFAT titers had declined 4-fold in three cows within 2 months of abortion.
These observations suggest that changes in paired serum samples from aborting cows
should not be relied upon to diagnose acute neosporosis. This outbreak was suggestive
of a point source exposure o f the herd to N. caninum.
4.16. Experimental neosporosis in cattle
Results of the two experimental studies indicate that N. caninum is a primary

pathogen and that generalized fetal infection can occur within 1 month after inoculation
of the dam (Dubey et al., 1992c; Barr et al., 1994a). In the first study, three multiparous
Jersey cows were inoculated IM and SC with three isolates of N. caninum obtained
from dogs (Dubey et al., 1992c). The three cows had been bred 129, 126 and 81 days
before N. caninum inoculation. Cow 1 was killed on Day 32 after N. caninum
inoculation. It had a live N. caninum infected fetus. Cow 2 aborted a macerated 210 g
fetus on 101 DAI and Cow 3 delivered a 500 g mummified fetus on 74 DAI. Fetuses
from Cows 2 and 3 were unsuitable for histologic examination. The fetus from Cow 1
had severe lesions in the brain and viable N. caninum was recovered both in cell culture
and in mice. Numerous N. caninum tachyzoites and one tissue cyst were demonstrated
histologically in sections of fetal brain. Lesions were also seen in placenta, lung and
spinal cord, but N. caninum was not found. Cows 2 and 3 were bred again and they
delivered clinically normal calves not infected with N. caninum.
In the second published study, six Simmental-cross heifers were inoculated IV and
IM with N. caninum tachyzoites of a bovine isolate (Barr et ai., 1994b). The heifers
were inoculated between 85 and 161 days of gestation. The three fetuses removed from
three heifers at 29, 30, and 31 DAI had confirmed generalized N. caninum infection. A
fetus from the 4th cow removed on 26 DAI was not infected. A fetus from the 5th heifer
died in utero and was mummified when removed 67 DAI; it was infected with N.
caninum. The 6th heifer delivered a live calf with a precolostral IFAT antibody titer of
1:20 480; N. caninum was not found in its tissues.
Because the natural postnatal mode of transmission of N. caninum in cattle is
unknown, it is not known when the placenta becomes infected. Therefore, it is
impossible to correlate results of these two experimental studies with natural infections.
Fetal gestational age at the time of N. caninum infection is probably important in the
pathogenesis. Fetuses infected before immunologic maturity (4-5 months) probably die
whereas those infected later in gestation develop less severe disease. Newborn calves
inoculated with N. caninum remained clinically normal (Dubey et al., 1996a). The level
of natural exposure to organisms may also be important in the clinical outcome.
4.17. Evidence that bovine Neospora of California is N. caninum
A group of researchers from the University of California at Davis, found protozoal

organisms associated with bovine abortions (Barr et al., 1990). Protozoa in bovine
fetuses reacted immunohistochemically with polyclonal serum from a rabbit immunized
with whole live N. eaninum (NC-1 isolate) and thus the organism was called Neospora
sp. (Anderson et al., 1991; Barr et al., 1991a). Conrad et al. (1993a) succeeded in
isolating Neospora from bovine fetuses and they characterized these isolates and
compared them with canine N. caninum isolates. Although these bovine isolates were
similar to N. caninum, they preferred to designate them as Neospora sp. rather than N.
caninum. In our opinion N. caninum is the only valid species of this genus. Differences
between the organisms found in canines and bovines are not greater than normally found
between strains of a species and are probably related to methods of examination and the
lack of knowledge of the full life cycle of N. caninum.
Structurally, tissue cysts of the canine parasite are the same as tissue cysts of the
bovine parasite. Tissue cysts in the bovine are often 1 to 3 p,m thick and rarely exceed
J.P. Dubey, D.S. Lindsay/ VeterinaryParasitology 67 (1996) 1-59 45
80 ~ m in diameter whereas tissue cysts in dogs may be as long as 107 p,m and the cyst
wall is up to 4 p,m thick (Dubey et al., 1988a). However, without knowing the duration
of infection, this comparison is not valid. Moreover, the host can influence the size of
the cyst. Barr et al. (1991b) ultrastructurally compared tissue cysts of the bovine parasite
with the canine parasite. They found tubulo-vesicles between bradyzoites and an unusual
membrane bound vesicle in bradyzoites thought to be associated with the bovine
parasite; it was later found in the canine parasite (Bjerk[ts and Dubey, 1991). In
summary, there are no differences in uitrastructure of cysts or bradyzoites between the
canine and bovine isolates of the parasite (Jardine, 1996).
UltrastructuraUy, cell culture-derived tachyzoites of the bovine isolates (Conrad et al.,
1993a) are identical with cell-culture derived tachyzoites of the canine isolates (Lindsay
et al., 1993). Conrad et al. (1993a) pointed out a few differences between cell
culture-derived tachyzoites of the bovine isolates from in vivo derived tachyzoites of the
canine isolates. However, this comparison is not valid because the ultrastructure of in
vivo derived bovine isolates was not compared. Moreover, the differences were minor.
For example Conrad et al. (1993a) found that the bovine isolates of Neospora divided in
cultures by endodyogeny whereby as many as four tachyzoites remained attached to
each other at the posterior end. Lindsay and Dubey (1989a) had earlier found similar
divisional process in preparations examined after cells had been scraped from the
monolayers. Fig. 1A to C in the present paper are from preparations where monolayers
were fixed first and scraped later. Therefore, the divisional process is similar in both
canine and bovine isolates. We would also like to point out that the divisional process in
in vitro cultured cells is not identical to those in in vivo fixed cells.
Antigenically, there were some differences between the bovine and canine isolates.
However, these differences can be explained by the difference in stages of the parasites
compared, preparation of antisera, and the immunohistochemical methods used. For
example, differences were found when antisera from different rabbits (immunized with
different antigenic preparations) were used. In retrospect, it was learned that N. caninum
shares several antigens with both T. gondii tachyzoites (Bjerk~s et al., 1994) and
bradyzoites (McAllister et al., 1996c), Moreover, different immunohistochemical proce-
dures do affect the results. For example, there is more cross reactivity between N.
caninum and T. gondii if tissues are digested with proteolytic enzymes. Lindsay and
Dubey (1989c) did not digest tissues whereas Barr et al. (1991b) digested tissues in
trypsin. The antibody titers in naturally infected cattle were remarkably similar irrespec-
tive of the source of N. caninura antigen, either from the canine isolate (NC-1) or the
bovine (BPA-1) isolate (Par6 et al., 1995b). Identical disease occurs in cattle experimen-
tally infected with canine (Dubey et al., 1992c) and bovine (Barr et al., 1994b) isolates.
Finally, Marsh et al. (1995) found only minor differences in the small subunit
ribosomal RNA gene sequences among three canine isolates and 4 bovine N. caninum
California isolates. However, techniques associated with reading the nucleotide sequence
gel s may account for some of the differences between sequences in GenBank (Marsh et
al., 1995). Stenlund et al. (1996) compared genetic and morphologic characteristics of
their Swedish isolate of N. caninura from a calf with the NC-1 isolate from a dog and
concluded that there was no difference between them, including the 16S-like rRNA and
the ITS-I.
46 J.P. Dubey, D.S. Lindsay / Veteriaary Parasitology 67 (1996) 1-59
5. Neosporosis in other animals

5.1. Goats
Abortion associated with N. caninum was reported in herds of pygmy goats from
California (Ban" et al., 1992) and Pennsylvania (Dubey et al., 1992a), and in dairy goats
from Costa Rica (Dubey et al., 1996b). In the report from California, two fetuses from
different herds were examined histologically. Tissue cysts were seen in lesions of
encephalitis from both fetuses.
The neosporosis-infected fetus from Pennsylvania came from a small farm-of pygmy
goats that had experienced abortions, stillbirth, and birth of weak kids. One fullterm
fetus was submitted for diagnosis. Lesions were seen mainly in the brain and especially
in the midbrain. The encephalitis was characterized by numerous microglial nodules
with minimal necrosis and severe perivascular infiltration of mononuclear ceils. Numer-
ous tissue cysts were seen and some tissue cysts were degenerating. Tachyzoites were
not seen (Dubey et al., 1992a).
The dairy goat fetus came from a farm in Heredia, Costa Rica (Dubey et al., 1996b).
The fetus was a 3 1 / 2 month gestational age and Saanen-Toggenberg cross. The goat
herd had experienced undiagnosed abortion problem. Lesions were found mainly in the
brain. Grossly, there was hydrocephalus and a hypoplastic cerebellum. The meningoen-
cephalitis was characterized by ventriculitis, gliosis, necrosis, mineralization and vasculi-
tis. An unusual feature of this case was the presence of large numbers of tissue cysts and
the rarity of tachyzoites. There was also myositis and tachyzoites were seen in muscles.
The doe of this fetus had an N. caninum IFAT titer of 1:800. Five of 77 other does from
this herd had N. caninum IFAT antibodies; cross reactivity with T. gondii was not
observed.
Because of their size, pygmy goats can be used as a model for bovine neosporosis.
Lindsay et al. (1995b) inoculated six pygmy goats with N. caninum tachyzoites at
different stages of pregnancy (Does 1 and 2: early gestation; Does 3 and 4: midgesta-
tion; and Does 5 and 6: late gestation). Doe 1 aborted two autolyzed N. caninum-in-
fected fetuses 30 DAI. Doe 2 had a fetus that died in utero and was resorbed. Doe 3
delivered two healthy kids; N. caninum was not found in their tissues, but was isolated
from placenta. Doe 4 delivered one live kid and one stillborn kid. Neospora caninum
was found in the tissues of the stillborn kid but not in tissues of the live kid. Does 5 and
6 inoculated late in pregnancy delivered weak kids that died shortly after birth but N.
caninum was not found in their tissues. All six does were bred again and they delivered
clinically normal kids not infected with N. caninum. All 6 does had persistent N.
caninum IgG antibody titers for several months. Unlike IgG titers, the IgM titers were
low ( < 1:400) and were short-lived (1-2 weeks after inoculation).
Adult dairy goats, like pygmy goats, inoculated with N. caninum did not develop
clinical signs, but developed high titer antibodies to N. caninum (Dubey et al., 1996a).
5.2. Sheep
There is only one report of congenital neosporosis in a lamb (Hartley and Bridge,
1975; Dubey et al., 1990b). The affected lamb was born weak, partially ataxic, and died
at 1 week of age. The most prominent findings were in the spinal cord. There was
unilateral reduction of gray matter in the ventral horn and focal cavitation. The myelitis
was associated with degenerating and intact N. caninum tissue cysts; tachyzoites were
not seen. It is noteworthy that this congenital defect was identical to that found in
Neospora-infected calves (Dubey et al., 1990a).
Sheep are highly susceptible to N. caninum infection. In a pilot experiment, two
pregnant ewes were inoculated either IV or IM with N. caninum tachyzoites (Dubey and
Lindsay, 1990b). Both ewes aborted dead twin lambs 25 and 26 DAI. Lesions were
similar in all four fetuses and were present in brain and spinal cord, muscles, and
placenta. Encephalitis was characterized by multiple foci of gliosis, hemorrhage, necro-
sis, perivascular cuffs and infiltration of mononuclear cells; tachyzoites were seen in
lesions. Myositis was seen in three fetuses and tachyzoites were seen in leg muscles of
one of these fetuses. There was placentitis, but N. caninum was not identified. Both
ewes developed high levels of N. caninum antibodies (Dubey et al., 1996a).
In a well-designed and executed study by McAllister et al. (1996b), 36 pregnant ewes
were inoculated IV with 1.7 l0 s (low dose) or 1.7 x 106 (high dose) tachyzoites of
the NC-2 and the NC-Liv isolates (equal numbers pooled) at 65, 90, or 120 days of
gestation. The stage of gestation, but not the dose, determined the outcome of preg-
nancy. All 12 ewes inoculated at 65 days of gestation aborted, 36 to 69 DAI. Of the 12
ewes inoculated at 90 days gestation, seven aborted, three had weak lambs and two had
clinically normal lambs. Of the 12 ewes inoculated at 120 days of gestation, none
aborted and all lambs born were clinically normal; tissue cysts were found in the brains
of six of 15 lambs euthanized at 90 DAI. Tissue cysts were also seen in the brain of I 1
of 29 aborted fetuses and one of four weak lambs. Lesions were seen in tissues of nearly
all aborted fetuses, even in autolyzed or mummified ones. Lesions were present in brains
(90% of 51), placentas (88% of 17), tongue or diaphragm (42% of 48), heart (22% of
46), liver (28% of 50) and lung (12% of 26) of fetuses or lambs. The inoculated ewes
remained clinically normal and N. caninum was not found in tissues of five of the ewes
necropsied. The ewes developed N. caninum antibodies following experimental infec-
tion. The IFAT titers of 24 ewes that were sampled on the day of infection were < 1:50
and all of them seroconverted ( > 1:400) by 3 weeks postinoculation. Fourteen ewes
developed low levels of T. gondii antibodies in the IFAT test following N. caninum
infection. The easy induction of abortion, birth of weak lambs, and infected but
clinically normal lambs in the sheep provides a good model for bovine neosporosis at a
reduced cost.
In another experiment, three 1-week-old lambs inoculated IV, SC, or IM with
1.5 x 10 6 N. caninum tachyzoites remained clinically normal indicating that postnatally
infected sheep are not likely to develop clinical neosporosis (Dubey et al., 1996a). All
three lambs developed high levels ( ~ 1:6400) of N. caninum IFAT antibodies that did
not react with T. gondii antigen in the dye test or MAT.
5.3. Horses
Neosporosis was first found in an equine fetus that was aborted in 1985 (Dubey and
Porterfield, 1990). The fetus was 2 months premature and came from an 8-year-old
Appaloosa mare Without any antibodies to T. gondii. Only lung tissue was available for
retrospective study. Numerous N. caninum tachyzoites were seen in pulmonic lesions.
Neonatal neosporosis was observed in a l-month-old female Quarter horse foal from
Wisconsin (Lindsay et al., 1996c). The foal had circled its mare since birth, tripped over
obstacles and could not find its mare if separated from her. Ophthalmologic-examination
indicated that the foal was blind in the left eye and had no or only minimal vision in the
right eye. The foal was euthanized because of poor prognosis. At necropsy, no gross
lesions were seen. Histologically, lesions in both eyes were severe, but no stages of N.
caninum were identified in the globes. A 25 X 24 /zm thick-walled tissue cyst of N.
caninum was identified in the muscles around 1 eye. The tissue cyst wall was 2 /zm
thick and bradyzoites were 1.5 /xm in diameter and had a subterminal nucleus. This
finding is unusual because it represents the first time that a N. caninum tissue cyst has
been definitively identified outside of the CNS. Sections of thalamus and caudal
hypothalamus contained lesions and thick-walled tissue cysts of N. caninum. The tissue
cysts reacted with rabbit anti-N, caninum antiserum, but not rabbit anti-Sarcocystis
neurona or rabbit anti-T, gondii antiserum in the IHC test.
An unusual case of visceral neosporosis was reported from a 10 year old Appaloosa
mare (Gray et al., 1996). The mare had history of weight loss and anemia. A complete
necropsy was performed. Lesions were confined to intestines and mesenteric lymph
nodes. The lymph nodes were grossly enlarged, had evidence of necrosis, hemorrhage
and thrombosis, and tachyzoites were seen in sections. The small intestine was grossly
hyperemic and edematous. Histologically, the lamina propria and submucosa of the
small intestine were infiltrated by neutrophils and lymphocytes, and there was mild
vasculitis; N. caninum tachyzoites were seen in lesions. This is the first reported case of
intestinal neosporosis in any animal species.
Neosporosis was diagnosed in an aged Pinto mare with a 1-day history of rear limb
paralysis, bizarre behavior, and masticatory difficulty (Daft et al., 1996). Lesions were
seen in the CNS, peripheral nerves, and the myocardium. There was muitifocal
non-suppurative encephalomyelitis and polyrediculoneuritis, and mild, muitifocal non-
suppurative myocarditis. Neospora tachyzoites and tissue cysts were demonstrated by
IHC in brain, spinal cord and peripheral nerves. The mare also had a pituitary adenoma
and presumed Cushing's disease, an acute compression fracture of the 9th thoracic
vertebra and focal mycobacterial pneumonia. Immune suppression from endocrine
disorder may have predisposed the mare to clinical neosporosis. This is the first report of
N. caninum tissue cysts in peripheral nerves in any host.
Marsh et al. (1996) recently found Neospora organisms in the brain and spinal cord
of a horse with neurological signs in California, USA. They isolated the parasite in cell
cultures inoculated with neural tissue of the horse.
5.4. Deer
Neosporosis was diagnosed in a 2-month-old female black-tailed deer (Odocoileus
hemionus columbianus) found dead in California, USA (Woods et al., 1994). Lesions
and N. caninum were found in sections of lung, liver, and kidneys. Tachyzoites were
most numerous in lung and were associated with interstitial pneumonia. The nephritis
was characterized by interstitial inflammation, tubular necrosis and tachyzoites were
seen in tubular epithelium and tubular lumina. In the liver, tachyzoites were within
hepatocytes, Kupffer cells and in sinusoids.
Neosporosis was recently diagnosed in a fullterm stillborn deer (Cervus eldi siamen-
sis) from the Paris Zoo, France (Dubey et al., 1996c). Neospora caninum tissue cysts
were found associated with nonsuppurative encephalitis. Neosporosis was thought to be
the main disease affecting the decline of this endangered species in the zoo.
5.5. Cats
There is no report of naturally occurring neosporosis in cats, although cats can be
easily infected with N. caninum experimentally (Dubey and Lindsay, 1989a; Dubey and
Lindsay, 1989c; Dubey et al., 1990e). Clinical neosporosis was induced in nursing,
weaned, and pregnant cats inoculated with N. caninum. In Experiment 1, two 3-day-old
kittens inoculated orally and SC with a total of 1 million tachyzoites became ill. One
kitten died 17 DAI and the other was killed when moribund 29 DAI; both kittens had
severe encephalomyelitis and myositis and N. caninum was found in lesions (Dubey and
Lindsay, 1989c).
In Experiment 2, two adult cats were inoculated to study transplacental neosporosis.
A 5-year-old cat inoculated SC with 2 million tachyzoites at 47 days of gestation gave
birth to a fullterm kitten that died of severe generalized neosporosis. The queen became
anorectic and was euthanized on the 3rd day after parturition. There was a macerated
fetus in its uterus. The queen was found to have generalized neosporosis with massive
metritis associated with numerous N. caninum tachyzoites. Why this queen and her
kitten developed such severe neosporosis (still not induced in any other adult animal
species) is perplexing. The presence of numerous tachyzoites and absence of other
recognizable causes suggests that the disease was primarily neosporosis (Dubey and
Lindsay, 1989a).
Another 7-month-old cat was fed infected cat brain containing tissue cysts. She
remained clinically normal and was bred naturally with a laboratory-raised male cat. She
gave birth to three apparently healthy fullterm kittens 174 days after feeding tissue cysts
(the gestation period in cats is approximately 55 to 60 days). One kitten was killed at 2
days of age; it was found to have histologically verified N. caninum associated lesions
in several tissues. Neither lesions nor N. caninum was found in tissues of the remaining
two kittens killed at 22 and 30 days of age. Results of this experiment indicated for the
first time that N. caninum can be transmitted congenitally from an animal infected
before pregnancy and that the infection can be transmitted orally by carnivorism of
infected tissues.
In Experiment 3, five 84-day-old cats were inoculated SC and orally with 1.5 million
N. caninum tachyzoites. Two cats were also given corticosteroids; one of these died 16
DAI and the other was euthanized 21 DAI because of severe generalized neosporosis.
Three cats not given corticosteroids remained clinically normal except for loss of weight
in one cat. These three cats were killed 55 DAI; N. caninum tissue cysts were seen in
the brain of one cat that had lost weight (Dubey et al., 1990e).
5.6. Monkeys
At present, there is no evidence that N. caninum infects humans. Experimentally,
congenital infections were produced in two rhesus macaques (Macaca mulatta) by Barr
et al. (1994a). Monkeys were inoculated IM and IV with a total, of 1.6 l07 N.
50 J.P. Dubey. D.S. Lindsay/ VeterinaryParasitology 67 (1996) 1-59
caninum tachyzoites of a bovine isolate at 43 days of gestation. The fetuses were

removed 67 to 70 DAI of the dams. Both fetuses had evidence of congenital neosporosis
characterized by multifocal necrotizing nonsupporative encephalitis and mild necrotizing
amnionitis. Neospora caninum tachyzoites were seen in lesions, were isolated in cell
culture, and Neospora-specific antibodies were found in blood of both infected fetuses.
Overall, the disease in monkeys had remarkable similarities to congenital toxoplasmosis
in humans (Barr et al., 1994a).
5.7. Foxes
Bjerk~s et al. (1984) inoculated a blue fox (Alopex lagopus) IM with brain
homogenate from a dog naturally infected with N. caninum. Active inflammatory
lesions and N. caninum-like tachyzoites were seen in sections of brain tissue of the fox
killed 10 weeks after inoculation. No other details were given.
5.8. Raccoons
Two raccoons (Procyon lotor) fed N. caninum infected tissues remained clinically
normal (Dubey et al., 1993). Oocysts were not seen in their feces after feeding N.
caninum infected tissues but both raccoons developed N. caninum antibodies (Dubey,
unpublished).
5.9. Coyotes
Neospora caninum IFAT antibodies were detected in five of 52 naturally-exposed

coyotes from Texas, USA. Antibody titers were low; three were positive at a 1:25
dilution, one at a 1:50 dilution and one at a 1:100 dilution (Lindsay et al., 1996b). Three
coyote pups raised in captivity from a seronegative dam were fed Neospora-infected
mouse brains. All three pups remained clinically normal and did not shed oocysts; all
three developed IFAT titers of 1:800 to N. caninum. There was no cross reactivity to T.
gondii antigen in experimentally-infected coyotes indicating specificity of the IFAT used
for coyotes (Lindsay et al., 1996b).
5.10. Mice
Neospora caninum is infective to mice and the mouse model is useful to study
biology, immunology, and chemotherapy. The development of clinical neosporosis
depends on the isolate of the mouse, strain and dose of N. caninum, and the treatment
given to mice. Outbred Swiss Webster adult mice do not develop clinical neosporosis,
but N. caninum can encyst in them (Dubey et al., 1988b). By varying the isolate of N.
caninum, the dose of tachyzoites, and the dose of corticosteroids clinical neosporosis
was induced in outbred Swiss Webster mice (Lindsay and Dubey, 1989d; Lindsay and
Dubey, 1990a, Lindsay and Dubey, 1990b, Lindsay et ai., 1991). The NC-I isolate was
more virulent to mice than the NC-2 and NC-3 isolates. Mice given two doses of 4 mg
of methylprednisolone acetate (MPA) and 1 105 tachyzoites developed generalized
neosporosis and generally died within 20 DAI. Mice given 2 mg of MPA developed
milder disease and often developed neurologic signs of neosporosis. In generalized
J.P. Dubey, D.S. Lindsay/ VeterinaryParasitology67 (1996) 1-59 51
neosporosis, tachyzoites were found in several tissues whereas in chronic neosporosis,

N. caninum was confined to the brain and spinal cord. Tissue cysts were found only in
the CNS and were first seen 21 DAI. Tissue cysts were < 50 /xm in diameter,
irrespective of the duration of infection.
Immunodeficient mice can develop severe neosporosis. Nude mice developed clinical
neosporosis after inoculation with NC-1 isolate tachyzoites (Yamage et al., 1996).
Knockout mice deficient in gamma interferon developed fatal neosporosis after IP
inoculation of 10000 NC-1 isolate tachyzoites (Dubey and Urban, unpublished, 1996).
Inbred B A L B / c mice are more susceptible to N. caninum infection than outbred
mice. Depending on the isolate and dose of N. caninum, B A L B / c mice developed
subclinical to clinical neosporosis (Lindsay et al., 1995a). The NC-1 isolate was more
virulent than the NC-3 isolate. B A L B / c mice infected with N. caninum develop
encephalomyelitis and tachyzoites were present in chronic lesions even up to 463 DAI
(Dubey, unpublished). Characteristic N. caninum tissue cysts in B A L B / c mice are rare.
Neospora caninum is transmitted congenitally in B A L B / c mice (Long and Baszler,
1996). Infection of dams with N. caninum 10 days before pregnancy, and 5, 10, and 14
days of gestation resulted in fetal death and resorption.
Transplacental transmission of N. caninum occurred also in outbred Swiss Webster
mice (Cole et al., 1995a). Congenital infection was seen in 11 of 13 litters and the
number of pups congenitally infected per litter was higher if dams were inoculated
during the first half of gestation. Lactogenic transmission and transmission during the
chronic stage were rare. Neosporosis has not been followed in congenitally infected
mice.
5.11. Rats
Sprague Dawley rats (Rattus norvegicus) were resistant to N. caninum infection

(Lindsay and Dubey, 1990c). Treatment with 2 or 4 mg of MPA was required to
produce clinical neosporosis in rats. Rats given 1 l0 s or more N. caninum tachyzoites
and 4 mg of MPA died of acute neosporosis involving liver, lungs and brain. Tissue
cysts were rare in rat tissues and a single cyst was seen in thebrain of a rat 17 DAI,
5.12. Gerbils
Three gerbils inoculated IP with naturally infected canine tissues became infected
with N. caninum (Cuddon et al., 1992). Numerous tachyzoites were found in the
peritoneal cavity of gerbils. However, organisms obtained from gerbils were not
infective to a second set of gerbils.
Acknowledgements
We thank D. Buxton, H.R. Gamble, M.C. Jenkins, N.C. Lally, M.M. McAllister, M.
Rommel, A.J. Trees, A. Uggla and W. Wouda, for their help in preparation of this
paper.
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veterinary

ELSEVIER Veterinary Parasitology 67 (1996) 1-59

A review of Neospora caninum and neosporosis

Keywords: Neospora caninum; Neosporosis;Tachyzoites, Tissue cysts; Abortion; Paralysis; Animals

1. Introduction and history

2. Structure and biology

* Corresponding author. Tel: 301-504-8128;fax: 301-504-9222;e-mail: JDUBEY@GGPLARSUSDA.GOV.

0304-4017/96/$15.00 Published by Elsevier Science B.V.

micronemes. The discrepancy in the number of rhoptries reported by different re-

2.2. Life cycle

2.3. In vitro cultivation

a Oocysts were not found in feces of any of these animals.

2.4. Host-parasite relationship

Neospora caninum is capable of producing grossly visible necrotic lesions in a few

proteins of approximately 30 KDa. Both of these proteins were recognized by N.

2.6. Molecular biology

There is a growing interest in studying N. caninum at the molecular level. Molecular

a Based on personal communication with authors.

Although neosporosis may resemble toxoplasmosis clinically, T. gondii and N.

2.8. Experimental treatment

Progress is being made in determining the susceptibility of tachyzoites of N. caninum

a All sulfonamides were given at 10 /.~g m l - t.

3.1. Seroprevalence in the general population

Only limited information is available concerning prevalence of N. caninum infection

3.2. Clinical signs

For antemortem diagnosis, clinical signs may be helpful. Ascending paralysis in

Ruehlmann et al. (1995) Vizsla 3 years Neurologic

by light microscopy. Tachyzoites should be looked for in C S F in cases o f hind limb

3.4. Reports of clinical neosporosis

3.6. Experimental infections

Historically, Thilsted and Dubey (1989) first reported N. caninum-like organisms in

Retrospectively, the earliest recorded case of neosporosis in cattle appears to be in a

4.2. Prevalence of abortion

a Earliest record of retrospective neosporosis abortion in cattle.

a Fetuses with confirmed N. caninum infection.

4.3. Clinical signs

4.4. Season and abortion

Neosporosis-induced abortions occur year round (Anderson et al., 1991; Tllurmond et

4.5. Abortion in dairy vs. beef cows

4.6. Age of the aborting cow

4.7. Age of the fetus

4.8. Epizootic or sporadic abortion

4.9. Repeat abortions

4.10. Fetal lesions and pathogenesis of abortion

( D u b e y et al., 1992c). T i s s u e cysts w e r e not reported in b o v i n e fetal tissues r e m o v e d

4.11. Diagnosis of bovine neosporosis fetal abortion

4.12. Neosporosis in congenitally infected calves

Neosporosis-infected calves may have neurologic signs, be underweight, unable to

a Calves had precolostral N. caninum antibodies.

4.13. Repeat congenital infection

Repeat congenital neosporosis is probably more common than repeat Neospora

4.14. Detection of N. caninum antibodies in cows

Detection of N. caninum-specific antibodies in the sera of cows can be useful in the

a Data from Barr et al. (1994b).

with an anti-mouse horseradish peroxidase conjugate to reveal the bound antibody. At an

4.15. Seroepidemiology of bovine neosporosis

4.16. Experimental neosporosis in cattle

Results of the two experimental studies indicate that N. caninum is a primary

4.17. Evidence that bovine Neospora of California is N. caninum

A group of researchers from the University of California at Davis, found protozoal