Isolation of Neospora caninum from dairy
zero grazing cattle in Israel
L. Fish, M. Mazuz, T. Molad, I. Savitsky, V. Shkap*
Division of Parasitology, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel
Received 11 June 2007; received in revised form 29 July 2007; accepted 1 August 2007
First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy
farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to
infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the
identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5
gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils
(Meriones tristrami) were inoculated intraperitoneally with 103to 105tenfold dilutions of subculture tachyzoites. The inoculated
gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no
neurological signs or deaths were seen during 4 months of observation.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Neospora caninum; Isolation; Cattle; Cell culture; Susceptible gerbils
Neospora caninum is an apicomplexan parasite
considered as a significant cause of bovine abortion
worldwide (Anderson et al., 2000; Dubey, 2003; Dubey
etal., 2007). Cattle that were seropositiveto N.caninum
were found to be 3–11 times more likely to abort than
1997; Davison et al., 1999a; Weston et al., 2005).
Although the N.caninumlife cyclehasbeen reproduced
by oral transmission between the definitive host (dog)
and cattle, transplacental infection from persistently
infected dams isknown to be the main route ofinfection
fetal transmission of tachyzoites from an infected dam,
across the placenta to the fetus (Barr et al., 1994;
Anderson et al., 1997; Maley et al., 2003, 2006; Dubey
et al., 2006). Although demonstration of specific
antibodies in the fetus is an important tool in diagnosis
of Neospora-associated abortion, a negative serological
result does not rule out infection (Dubey and Schares,
2006). Therefore, isolation or demonstration of N.
caninum in the tissues of an aborted fetus is considered
as the most accurate and reliable diagnostic evidence
for N. caninum-induced abortion. On the other hand,
the isolation procedure may be negative due to the
unbefitting source of the fetal material. In addition, it is
well documented that prevalence of fetal infection
reflects the prevalence of maternal infection, mainly
resulting in non-fatal infection of the fetuses (Dubey
et al., 2007), therefore, demonstration of N. caninum in
fetal tissues supports the diagnosis, but may not be
definitive (Dubey and Schares, 2006). More than 45%
Veterinary Parasitology 149 (2007) 167–171
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fax: +972 3 9681678.
E-mail address: firstname.lastname@example.org (V. Shkap).
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seropositivity to N. caninum has been found in dams of
dairy cattle in Israel (Reske, 2000), and 41% of fetuses
were demonstrated to be positive serologically or by
PCR (Fish et al., 2004). Notably, Israel dairy herds are
kept on zero grazing, and the Neospora-associated
abortions are of an endemic pattern. Here we report on
the first isolates of N. caninum obtained from two
aborted fetuses from dairy herds located in southern and
northern Israel and kept all year around on zero grazing.
2. Materials and methods
2.1. Aborted fetuses
Two naturally aborted fetuses from the second and
third trimesters of gestation were collected from two
geographically separated dairy herds. Both herds
consist of Israeli Holstein cattle that are barn-housed
year-round, and among which breeding is exclusively
by artificial insemination. Both farms are closed units
which raise their own cows and do not purchase from
outside. Neosporosis is endemic on these farms. The
aborted fetuses from the southern part of Israel, i.e., the
Negev, and from the northwestern part of the country
were designated NcIs491 and NcIs580, respectively.
The aborted fetuses were kept chilled for 24 h pending
Fetal fluid collected from the pleural cavity from
both fetuses was centrifuged at 1000 ? g for 15 min,
and tested for specific N. caninum antibodies by the
immunofluorescence antibody test (IFA) (Shkap et al.,
2002). The slide IFA antigens were prepared from NC1
at which the whole parasites showed bright fluorescence
was considered as the endpoint titer (Pare et al., 1995).
2.3. Isolation of N. caninum in cell culture
fetal brains were removed aseptically and placed into
phosphate-buffered saline (PBS) supplemented with
penicillin (600 IU/ml) and streptomycin (300 mg/ml). A
portion of the brain material was washed three times in
PBS, cut into small pieces, homogenized with a tissue
grinder in 5 ml of Leibovitz-15 (L15) culture medium
(Biological Industries, Bet Haemek, Israel) supplemen-
ted with penicillin (200 IU/ml), streptomycin (100 mg/
ml) and mycostatin (75 IU/ml) (BioLab, Israel). After
centrifugationat70 ? gfor5 min,aconfluentmonolayer
of cells was loaded with 0.5 ml of the supernatant. The
cultures were maintained at 37 8C under 5% CO2. After
24 h the growth medium was replaced with the
maintenance medium, consisting of L15 and McCoy
medium and 2% heat-inactivated horse serum and
antibiotic/anti-mycotic solution. The maintenance med-
ium was subsequently changed every 2–3 days. The
subculture process was performed once a week.
2.4. Nested polymerase chain reaction (nPCR)
The nPCR was applied for the detection of parasite
DNA in brain tissues, and in Vero cell cultures
inoculated with the suspect brain material. The DNA
was extracted with the genomic DNA purification kit
(Puregene, Gentra, Minneapolis, MN, USA). The nPCR
on cell culture material was performed weekly. The
primers for the nPCR were based on published
sequences of the Nc5 gene of the NC1 isolate (GenBank
accession no. X84238). The external primers designed
fromthe sequence analysis were:
CTGCTGACGTGTCGTTGTTG-30, position 476 and
reverse, 50-CATCTACCAGGCCGCTCTTC-30, posi-
tion 1014. The inner primers were: forward, 50-
position 910. The PCR assays were performed in a
final volume of 40 ml and contained 5–10 mg of
genomic DNA, 100 ng of each primer and 2? Reddy
Mix PCR Master Mix (Abgene) containing 0.3125 units
Thermoprime Plus Polymerase, 18.75 mM Tris–HCl
(pH 8.8), 0.375 mM MgCl (NH4)2SO4and 0.05 mM
dNTP mix. The cycling protocol comprised an initial
3 min denaturation at 95 8C, followed by 30 cycles of
30 s denaturation at 94 8C, and 30 s of annealing at
56 8C, followed by 40 s elongation at 72 8C, with final
extension for 5 min at 72 8C. For nested amplification
1 ml of the primary PCR product was used as a template
was as above, except that the annealing was at 57 8C
followed by 30 s elongation in each cycle. In the nPCR,
DNA preparations from NC1 isolate, from serologically
negative fetuses, from Toxoplasma gondii, and from
Besnoitia besnoiti were included as positive and
negative controls. The amplified products were visua-
lized in a 1.5% agarose gel stained with EtBr.
2.5. Inoculation of experimental animals
The Animal Welfare Committee of the Kimron
Veterinary Institute approved all experiments per-
formed with small animals. Three 6 months old gerbils
L. Fish et al./Veterinary Parasitology 149 (2007) 167–171 168
(Meriones tristrami shawii) were inoculated with
tachyzoites of the NcIs491 isolate obtained from
passage six of the infected Vero cells. The infected
cell material was passed through a 27 G needle and
then centrifuged at 70 ? g for 5 min to obtain parasites
separated from cell debris (Baszler et al., 1999).
Tachyzoites, counted in a hemocytometer, were
inoculated into gerbils, subcutaneously. The gerbils
were bled from the retro-orbital sinus on the day of
inoculation, and on days 4 and 84 thereafter for the
detection of specific antibodies (Shkap et al., 2002;
Pipano et al., 2002).
Specific N. caninum antibodies were detected in
the fluids of both infected fetuses, with the IFA end
point dilution of 1:640 (Table 1). The nPCR amplified
a specific N. caninum fragment of 299 bp from the
brain DNA of the fetus 491, but no amplification was
obtained with DNA from fetus 580 (Fig. 1, Lanes 1 and
5). Tachyzoites of N. caninum were first observed
microscopically in Vero monolayers on days 30 and 32
after infection with brain material from fetuses 491 and
580, respectively. Amplification of 299 bp specific
fragments was obtained by nPCR (Fig. 1) with genomic
DNA that had been extracted from cultures 5 days
earlier, namely, on days 25 and 28 after cultures were
infected, although at that point no parasites were visible
under the microscope. No amplification was obtained
with DNA from the taxonomically related T. gondii or
B. besnoiti. Similar results were obtained in the IFA
assay with either NC1- or NcIs491-based antigen tested
with known positive and negative serum samples. In the
inoculated gerbils seroconversion was observed 28 days
after infection, and a similar reciprocal titer of 1:4096
was maintained at day 84 post-infection (Table 1).
Characteristic neurological signs in the infected gerbils
were not observed during 4 months of observation
High seroprevalence of N. caninum in Israeli dairy
herd dams and aborted fetuses has been previously
reported (Shkap et al., 2002; Fish et al., 2004), however
no local isolates were obtained. Despite the high
seroprevalence, it is well established that the efficiency
and especially on the state of the fetus (Dubey, 2003).
Vianna et al. (2005) reported that not all isolates can
grow or maintained in cell culture. Isolation methods
are completely dependent on the presence in the fetus
tissues of viable parasites capable of penetration and
propagation in host cells. Despite serious difficulties,
therewere manyreports,from various parts oftheworld
of successful isolation of N. caninum (Conrad et al.,
1993; Davison et al., 1999b; Kim et al., 2000; Canada
The fetal fluid of both fetuses showed IFA reciprocal
titer of 1:640 when subjected for parasite isolation,
which presented a highly significant indication of N.
caninum infection, as according to Dubey (2003) an
L. Fish et al./Veterinary Parasitology 149 (2007) 167–171169
Fig. 1. Neospora caninum nPCR amplification of DNA extracted
from fetal brains and infected cell cultures. Lane 1: DNA from fetus
brain 580; Lanes 2–4: DNA from culture infected with brain material
from fetus 580 on days 18, 25 and 28, respectively; Lane 5: DNA
brain material from fetus 491 on days 18, 25 and 28, respectively;
Lane 9: DNA from a non-infected brain; Lane 10: DNA from non-
infected Vero cells; Lane 11: DNA from culture infected with NC1 N.
caninum strain; Lane 12: 1 kb marker.
Infection of cell culture and gerbils with Neospora caninum
Brain tissues Culture Positive (on day)Inoculated with (survived) IFA titer (on day)
491 1:64030 Positive 21103(2/2)
1:1024 (28 and 84)
5801:64032 Negative21Not done
aEnd point dilution in immunofluorescence antibody assay.
bNested polymerase chain reaction.
cOne animal died of non-relevant infection.
end-point dilution of 1:25 is regarded as specific for
Neospora infection. Despite negative PCR of one of the
fetuses we proceeded with the isolation procedure by
blind sub-passages of inoculated cell cultures, followed
by examinations of these cultures by nPCR. The N.
caninum parasites of two Israeli isolates were first
detected in culture after a month, they had similar
growth rates, although direct quantitative analyses have
not yet been undertaken. In isolation procedures
reported elsewhere, the period between introduction
of suspicious material to cell culture and first detection
of N. caninum in cultures varied between 5 days
(McInnes et al., 2006) and 9.5 weeks (Pastusiak et al.,
2005). According to Davison et al. (1999b) the earliest
observation of parasites in culturewas after more than 1
month, and it was found to be directly associated with
the number and viability of parasites used for the initial
infection. The nPCR applied in the present study was
used to confirm N. caninum infection, both in the brain
tissues of seropositive aborted fetuses and in cell
cultures. Notably, brain tissue of one of the fetuses was
nPCR negative, whereas another tissue sample from the
same brain yielded viable parasites capable of infecting
cell culture in vitro, which was consistent with the
discrepancies among the findings of various methods
for N. caninum isolation, when samples were taken
from non-infected portions of tissues.
A limited number of gerbils were injected. However
following inoculation of tachyzoites into of susceptible
gerbils, high level of specific antibodies was produced
(reciprocal titer of 1:4096), with no characteristic
neurological signs observed during 4 months of
observation. Although a more robust analysis would
include unnecessary sacrificing of more animals, the
results were quite clear and did not justify using more
It has been reported that isolates differ in their ability
to infect susceptible laboratory animals. Gerbils that
were infected with the same dose of culture-grown NC1
strain tachyzoites developed neuromuscular symptoms
or died (Pipano et al., 2002), whereas it appears that the
natural pathogenicity of the Israeli isolate differed from
the NC1 strain, as no clinical signs were produced in the
same animal model with comparable number of
parasites inoculated. Knowledge on strain variations
pathogenicity is limited. Multilocus microsatellite
analysis of nine Neospora in vitro cultured isolates
from various hosts and geographic regions revealed
distinct genetic profiles with 12 out of 13 markers
analyzed (Regidor-Cerrillo et al., 2006), but no
association was found between genetic similarities
pertained to either host origin, or location of the isolate.
Comparative analysis of six isolates showed significant
variation in the in vitro growth rate, as assessed by
3[H]uracyluptake, and geneticvariations were found by
RAPD-PCR (Schock et al., 2001), although no
differences were revealed by Western immunoblotting.
Marked differences in pathogenicity for mice, between
NC-Liverpool and NC-SweB1 isolates were demon-
strain produced severe clinical signs, including dis-
coordination, paralysis and loss of weight, the NC-
SweB1 isolate produced milder signs observed in a
smaller proportion of inoculated mice over the same
In the light of low pathogenicity for susceptible
gerbils observed, the Israeli strains need broader
studies, and this for development of vaccine based on
naturally low virulent strains.
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