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

www.elsevier.com/locate/vetpar

Veterinary Parasitology 149 (2007) 167–171

Abstract

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 103 to 105 tenfold 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

1. Introduction

Neospora caninum is an apicomplexan parasite

considered as a significant cause of bovine abortion

worldwide (Anderson et al., 2000; Dubey, 2003; Dubey

et al., 2007). Cattle that were seropositive to N. caninum

were found to be 3–11 times more likely to abort than

seronegative animals (Thurmond et al., 1997; Pare et al.,

1997; Davison et al., 1999a; Weston et al., 2005).

Although the N. caninum life cycle has been reproduced

by oral transmission between the definitive host (dog)

and cattle, transplacental infection from persistently

infected dams is known to be the main route of infection

(Dubey, 2003). Infection of the fetus occurs by materno-

* Corresponding author. Tel.: +972 3 9681758;

fax: +972 3 9681678.

E-mail address: shkapv@int.gov.il (V. Shkap).

0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2007.08.009

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%

L. Fish et al. / Veterinary Parasitology 149 (2007) 167–171168

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

examination.

2.2. Serology

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

isolate and from the NcIS491 isolate. The highest dilution

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

Tissue samples of about 10 g from various parts of the

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

centrifugation at 70 � g for 5 min, a confluent monolayer

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

from the sequence analysis were: forward, 50-CTGCTGACGTGTCGTTGTTG-30, position 476 and

reverse, 50-CATCTACCAGGCCGCTCTTC-30, posi-

tion 1014. The inner primers were: forward, 50-GCGTCAGGGTGAGGACAGTG-30, position 631,

and reverse, 50-CTCTCCGTTCGCCAGCAGTG-30,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)2SO4 and 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

in a total volume of 40 ml. The cycling protocol in nPCR

was as above, except that the annealing was at 57 8Cfollowed 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) 167–171 169

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

extracted from brain 491; Lanes 6–8: DNA from cultures infected with

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.

(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).

3. Results

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

period.

Table 1

Infection of cell culture and gerbils with Neospora caninum

Fetus IFA titera Culture positive

(on day)

Nested PCRb

Brain tissues Culture Posi

491 1:640 30 Positive 21

580 1:640 32 Negative 21

a End point dilution in immunofluorescence antibody assay.b Nested polymerase chain reaction.c One animal died of non-relevant infection.

4. Discussion

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

of parasite isolation depends on the sampling procedure,

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,

there were many reports, from various parts of the world

of successful isolation of N. caninum (Conrad et al.,

1993; Davison et al., 1999b; Kim et al., 2000; Canada

et al., 2002; Pastusiak et al., 2005; McInnes et al., 2006).

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

Gerbils

tive (on day) Inoculated with (survived) IFA titer (on day)

103 (2/2) 1:1024 (28 and 84)

104 (1/2)c 1:4096 (21)

105 (2/2) 1:4096 (21)

Not done

L. Fish et al. / Veterinary Parasitology 149 (2007) 167–171170

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 culture was 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

gerbils.

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

among N. caninum strains (isolates) with respect to their

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 by3[H]uracyl uptake, and genetic variations 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-

strated by Atkinson et al. (1999). Whereas the Liverpool

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

period.

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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