The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina.

Abstract

The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous bovine abortions in Argentina. Based on histopathological results, 70 presumptive cases of apicomplexan protozoal abortion from a total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999 to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available. Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in any aborted bovine fetus.

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The role of Neospora caninum and Toxoplasma gondii
in spontaneous bovine abortion in Argentina
D.P. Moorea,*, J. Regidor-Cerrillob, E. Morrellc, M.A. Posoc,
D.B. Canoc, M.R. Leundac, L. Linschinkyc, A.C. Odeo ´nc,
E. Odriozolac, L.M. Ortega-Morab, C.M. Camperoc
aConsejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (CONICET), CP C1033AAJ Buenos Aires, Argentina
bSALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid,
Ciudad Universitaria s/n, 28040 Madrid, Spain
cEstacio ´n Experimental Agropecuaria, Patologı ´a Veterinaria, Instituto Nacional de Tecnologı ´a Agropecuaria (INTA),
CC 276, 7620 Balcarce, Argentina
Received 10 April 2008; received in revised form 19 June 2008; accepted 20 June 2008
Abstract
The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous
bovine abortions in Argentina.Based on histopathological results, 70 presumptive cases of apicomplexanprotozoal abortion from a
total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999
to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by
immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was
extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available.
Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there
was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in
any aborted bovine fetus.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Argentina; Bovine fetuses; Histopathology; IFAT; IHC; Neospora caninum; Nested-PCR; Toxoplasma gondii
1. Introduction
Neospora caninum is an apicomplexan protozoa
which causes reproductive failure in cattle worldwide,
includingArgentina(Moore,2005;Dubeyetal.,2007).
Initial studies in Argentina detected N. caninum-
seropositive individuals among aborted cows (Ventur-
ini et al., 1995). IHC has also been used to confirm the
presence of the parasite in aborted bovine fetal tissues
(Campero et al., 1998). The prevalence of specific
antibodies varied from 4.7% of 400 beef cows
and 16.6% of 1048 dairy cows lacking a history of
reproductive losses (Moore et al., 2002). Similarly, in
animals with a history of reproductive losses there was
a lower prevalence of specific antibodies in beef cows
(18.9% of 216) compared with dairy cows (43.1% of
750) (Moore et al., 2002).
T.gondiiisaprotozoanparasiteinfectinghumansand
other warm-blooded animals. Recently, it was reported
that 91% of 90 Argentinean beef cattle were positive for
www.elsevier.com/locate/vetpar
Available online at www.sciencedirect.com
Veterinary Parasitology 156 (2008) 163–167
* Corresponding author.
E-mail address: pmoore@balcarce.inta.gov.ar (D.P. Moore).
0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2008.06.020

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the parasite as determined by indirect fluorescent
antibody test (IFAT). T. gondii infection has also been
confirmed by PCR (More ´ et al., 2008). Nevertheless, T.
gondii has not yet been linked to bovine abortion in
Argentina.
The aim of this study was to investigate the role of N.
caninum and T. gondii infections in spontaneous bovine
abortions in Argentina.
2. Materials and methods
2.1. Clinical samples and data collection
Whole specimens or tissues from 666 bovine fetuses
submitted for routine abortion diagnosis from 1999 to
August of 2007 were used in this study. The fetuses
originated from the humid pampas, which is the main
cattle-producing area of Argentina and includes the
province of Buenos Aires, north of the Rı ´o Negro, east
of La Pampa, south of Santa Fe, Co ´rdoba, and south of
Entre Rı ´os.
Diagnosis for infectious agents causing abortion was
performed by various laboratory methods as described
by Campero et al. (2003). Based on histopathological
results, 70 presumptive cases of apicomplexan proto-
zoal abortions were included in this study.
For each fetus submitted for diagnostic testing, the
cattle production system (beef or dairy), age of the fetus
and pattern of abortion were recorded. The pattern of
abortions was defined as epidemic if >10% of the cows
were at risk of abortion within 6 weeks, or endemic if
the cows persisted in the herd for several months or
years (Wouda et al., 1999).
2.2. Histopathology and IHC for N. caninum
Fetal tissues were fixed in 10% neutral buffered
formalin, embedded in paraffin, and stained with hema-
toxylin and eosin (H&E) for routine histologic examina-
tion. All tissues with microscopic lesions compatible
with those produced by apicomplexan protozoan (non-
suppurative meningoencephalitis, myocarditis,hepatitis,
miositis and placentitis) were processed by IHC.
IHC was performed using the avidin biotin complex
(Vector, Peroxidase Elite ABC PK-601) method for N.
caninum (Lindsay and Dubey, 1989). Briefly, a second
set of paraffin sections were mounted on positive-
charged glass slides (Probe-On Plus; Fisher Scientific)
and processed for IHC as previously described with an
automated capillary action immunostainer (Micro-
Probe TM; Fisher Scientific). Sections were dehy-
drated, enzymatically treated with 0.4% pepsin (Sigma
ChemicalCo.,St.Louis,MO)for antigenretrieval,and
incubated with 0.5% casein (Sigma Chemical Co., St.
Louis, MO) to block nonspecific immunoglobulin
binding. The primary antibody was anti-N. caninum
hyperimmune polyclonal rabbit serum (kindly pro-
vided by Dr. M. Anderson from UC Davis, US) diluted
1:200. Immunostaining was visualized with amino-
ethylcarbazol substrate (Dako Inc.), and sections were
counterstained with Mayer’s hematoxylin (Sigma
Diagnostics, St. Louis, MO) and examined micro-
scopically. Positive control tissue consisted of for-
malin-fixed brain tissue from mice experimentally
inoculated with the NC-1 strain of N. caninum.
Negativecontrols were samples testedwithout primary
antibody but with a similar dilution of normal rabbit
serum.
2.3. Testing for antibodies to N. caninum in
thoracic-abdominal fluids
Thoracic–abdominal fluids from 55 of 70 aborted
fetuses were available for examination for antibodies
specific to N. caninum by IFAT. A titer of ?1:25 was
considered positive (Moore et al., 2002).
2.4. DNA extraction from fresh and formalin-fixed,
paraffin-embedded tissue
DNA was extracted from formalin-fixed paraffin-
embedded tissues from 53 cases and from frozen tissues
from 17 cases, collected from 2004 to the present. Fresh
fetal samples were collected at necropsy and stored at
?80 8C until tested (Collantes-Ferna ´ndez et al., 2006b).
When frozen tissues were available, formalin-fixed
paraffin-embedded tissues were not tested by nested-
PCR. CNS tissue was processed from 95 samples from
50 cases of abortion. Tissues from 20 cases [heart (19),
liver (11), muscle (3) and placenta (3)] were processed
when CNS tissue was not available.
For formalin-fixed tissues, ten 12-mm paraffin
sections were cut with a standard microtome. The
microtome bladewas cleaned between blocks with 70%
ethanol to prevent cross-contamination. Excess paraffin
was removed with sterile forceps, and the tissue was cut
into smaller pieces using a scalpel. Forty milligrams of
tissue were used for DNA extraction from formalin-
fixed tissues. Tissues were placed in 1.5-ml micro-
centrifuge tubes and dewaxed with xylene and ethanol
washes; 20 mg of tissue were used for DNA extraction
from fresh tissues. Genomic DNA from frozen and
formalin-fixed paraffin-embedded tissues was extracted
using a commercially available kit (Real Pure, Durviz,
D.P. Moore et al./Veterinary Parasitology 156 (2008) 163–167164

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Paterna, Spain) according to the manufacturer’s
instructions. The concentration of DNAwas determined
by spectrophotometric analysis at A260/280 and all
the samples were diluted to a final concentration of
60 ng/ml. DNA samples were stored at ?20 8C until
PCR analysis.
2.5. PCR amplification of N. caninum and T. gondii
ribosomal RNA
In order to exclude the possibility of false-negative
PCR results due to poor-quality DNA or the presence of
nonspecific PCR inhibitors in the clinical tissue
samples, conventional PCR for the amplification of
28S rRNA gene was performed using the primers
described by Collantes-Ferna ´ndez et al. (2002).
Neospora DNAwas detected by a nested-PCR on the
internal transcribed spacer (ITS1) region with four
oligonucleotides as described by Buxton et al. (1998).
In addition, a single-tube nested-PCR assay as
described by Hurtado et al. (2001) was performed to
detect T. gondii DNA on all samples. Positive (purified
N. caninum or T. gondii tachyzoite DNA) and negative
controls (DNA from normal bovine brain, paraffin
control, and double-distilled water) were included in
each PCR.
Secondaryamplification productswerevisualizedby
1.8% agarose gel electrophoresis and ethidium bromide
staining under UV light.
2.6. Analysis of data
Agreement between the different diagnostic tech-
niques was estimated by calculating kappa values (K-
value) (Thrusfield, 1995) with the statistical program
Win Episcope (Win Episcope, 2000). K-values bet-
ween 0.4 and 0.6 indicate moderate agreement,
values between 0.6 and 0.8 indicate good agreement,
and values between 0.8 and 1.0 indicate very good
agreement.
Associations between detection of N. caninum
infection by the different diagnostic techniques and
cattle production system (beef or dairy), pattern of
abortion,andfetalagewereanalyzed bythex2orFisher
tests with the STATGRAPHICS Plus 5.1 software (Stat
Point, Herndon, Virginia).
Forallthetests, asignificance levelof0.05wasused.
3. Results
Seventy of 666 cases of bovine abortion showed
histopathological lesions consistent with protozoal
abortion, and were negative for bacterial and viral
agents of abortion. In 49 of these 70 cases (70%),
Neospora antigens were identified by IHC; 31 of 55
available fetal fluids (56.4%) had antibodies specific for
N. caninum. Neospora DNA was detected in 17 of 17
fetuses analyzed from frozen tissues and in 17 of 53
fetuses from formalin-fixed paraffin-embedded tissues.
N.caninuminfectionwasdocumented byatleastone
diagnostic technique used (IFAT, IHC or/and nested-
PCR) in 66 of the 70 cases (9.9% of 666 fetuses). N.
caninum infection was confirmed by all three techni-
ques in 9 of 70 bovine fetuses (12.8%). N. caninum
infection was detected by at least two techniques in 30
fetuses (42.8%). Positive results were obtained from
one diagnostic technique in 27 cases (38.6%). In 4 cases
N. caninum infection was not identified. Poor agree-
ment was observed between the IFAT and IHC
techniques (k = ?0.038), between IFAT and PCR
(k = ?0.003) and between IHC and PCR (k = ?0.158).
AcomparisonofPCRresultsfromdifferenttissuesis
shown in Table 1.
These 70 cases included 37 fetuses obtained from
beef herds and 24 fetuses obtained from dairy herds
(this information was missing for nine specimens).
Thirty-two fetuses couldbeclassified asassociated with
an epidemic or endemic pattern of abortion. Diagnostic
results for N. caninum infection are summarized in
Table 2.
The percentage of N. caninum infections detected by
the three diagnostic techniques did not differ signifi-
cantly (P > 0.05 by x2or Fisher tests).
For T. gondii infection, nested-PCR was negative for
all fetal samples.
4. Discussion
In the present work definitive diagnosis of N.
caninum and T. gondii as causes of bovine abortion
was attempted in selected cases of abortion with
histological lesions suggestive of protozoal infection
(Ortega-Mora et al., 2006). N. caninum infection was
confirmed in 66 of 70 (>90%) cases of spontaneous
D.P. Moore et al./Veterinary Parasitology 156 (2008) 163–167165
Table 1
Results from different tissues by nested-PCR
Fetal tissueNumber examinedNumber positive%
CNS
Liver
Heart
Placenta
Muscle
95
11
19
3
3
2324.2
54.5
10.5
33.3
0
6
2
1
0

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bovine abortion analyzed for the presence of protozoan
antigen, DNA and/or specific antibodies.
The higher sensitivity of IHC over PCR for detecting
N.caninuminfectionsinthisstudycontrastswithresults
reported by other authors (Baszler et al., 1999; Maanen
et al., 2004). Unfortunately false positive diagnoses are
frequently observed with IHC (Reitt et al., 2007), and it
may not be a reliable diagnostic test for N. caninum
infection when used by itself.
Itisnoteworthythat100%of17freshfrozenbutonly
32% of 53 formalin-fixed paraffin-embedded samples
were positive by PCR. In contrast, only 11 of the 17
fresh frozen samples were positive by IHC (data not
shown). Although Baszler et al. (1999) reported a
sensitivity of 77 and 100% for fresh brain and formalin-
fixed, paraffin-embedded brain tissues, respectively, the
poor quality of DNA extracted from formalin-fixed,
paraffin tissues could explain the decreased number of
positive samples. In addition to the better results
obtained from fresh frozen samples in our work, DNA
extraction isnot onlycheaper,butalso faster when fresh
frozen tissues are available.
The optimal tissues for diagnosing Neospora-asso-
ciated abortions are fetal brain, heart and liver (Dubey
and Schares, 2006). In agreement with Wouda et al.
(1997), the parasite was more frequently identified by
IHC in the liver than in the heart. We also found a higher
proportion of positive nested-PCR in liver tissue
compared to brain tissues. However, 95 brain samples
from 50 cases were tested while only 11 liver specimens
were available. There is no doubt that CNS tissue is
the best to identify N. caninum (Wouda et al., 1997;
Collantes-Ferna ´ndez et al., 2002; Dubey and Schares,
2006).
IFAT detected a smaller percentage of N. caninum
infections in comparison to IHC. This could be due to
the limited immune competence in younger fetuses (<5
months gestational age) or the short interval between
infection and fetal death, or the degradation of fetal
immunoglobulins in the examined fluids (Ortega-Mora
et al., 2006).
Kappa analysis showed limited agreement among
the diagnostic techniques used in this work (IFAT, IHC
and nested-PCR). Thus, these results emphasize the
necessity of combining these complementary techni-
ques to enhance detection of N. caninum infections in
aborted fetuses (Pereira-Bueno et al., 2003).
Although significant differences were not detected
among the diagnostic techniques, the better perfor-
mance of PCR in specimens from herds with an
epidemic pattern of abortions agrees with the recent
findings of Collantes-Ferna ´ndez et al. (2006a). On the
other hand, a higher proportion of IHC positive fetuses
from cases with an endemic pattern of abortions may
have been due to a high rate offalse positives using IHC
(Thurmondetal.,1999).Alternatively,ahighnumberof
positive-IHC tissue samples have been observed in
fetuses in the second trimester compared to the last
trimester (Collantes-Ferna ´ndez et al., 2006b).
Although, T. gondii infection was recently detected
in adult beef cattle (More ´ et al., 2008), none of the 70
fetuseswerepositivebynested-PCRinthisstudy.These
findings are in agreement with a study from Switzerland
(Reitt et al., 2007), but T. gondii infection has been
reported by other groups (Gottstein et al., 1998; Sager
et al., 2001), warranting further studies of the role of T.
gondii as a cause of abortion in cattle in Argentina.
In conclusion, aborted fetuses diagnosed by histo-
pathology as having a protozoal etiology were inves-
tigated by IFAT, IHC and PCR. N. caninum infection
was confirmed in 9.9% of 666 specimens sugges-
ting that it is a frequent cause of bovine abortion in
Argentina.
Acknowledgements
We thank Antonio Rodrı ´guez-Bertos from the
Faculty of Veterinary Sciences, Complutense Univer-
sity of Madrid, and Germa ´n Canto ´n from INTA
Balcarce for their excellent technical assistance. DP
Moore thanks Dr Douglas Hodgins (Ontario Veterinary
College/U of Guelph, Canada) for critical review and
helpful comments. This work was funded by Agencia
D.P. Moore et al./Veterinary Parasitology 156 (2008) 163–167 166
Table 2
Diagnostic results for Neospora caninum by histopathology, by
indirect fluorescent antibody test (IFAT), by immunohistochemistry
(IHC) and by nested-PCR according to cattle production system (beef
or dairy), pattern of abortion and fetal age
Diagnostic technique percentage (analyzed)
H&E+ IFAT+ IHC+PCR+
Cattle production system
Beef cattle
Dairy cattle
Undetermined
8.3 (442)
13.7 (175)
18.3 (49)
61.1 (36)
47.4 (19)
–
62.1 (37)
83.3 (24)
66.7 (9)
54 (37)
41.7 (24)
44.4 (9)
Pattern of abortion
Epidemic
Endemic
Undetermined
NRa
NR
NR
61.5 (13)
52.6 (19)
56.5 (23)
64.3 (14)
77.2 (22)
67.6 (34)
57.4 (14)
45.4 (22)
47.0 (34)
Fetal age
2nd trimester
3rd trimester
NR
NR
50 (38)
64.7 (17)
76.4 (51)
52.6 (19)
47 (51)
57.8 (19)
Total10.5 (666) 56.3 (55)70 (70)48.5 (70)
aNot recorded.

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Nacional de Promocio ´n Cientı ´fica y Tecnolo ´gica,
FONCyT, PICT 31681, INTA (AESA 3597) (Argen-
tina) and Programa de Creacio ´n y Consolidacio ´n de
Grupos de Investigacio ´n Universidad Complutense-
Comunidad de Madrid (CCG06-UCM/AGR-1283).
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