Detection of Toxoplasma gondii and Neospora caninum antibodies in Wild Boars (Sus scrofa) in Eastern Turkey

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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Balkaya, I. 28
INTRODUCTION
Toxoplasma gondii is one of the most important zoonotic
protozoa that infect a variety of birds and mammals, includ-
ing humans. Felids are the defnitive hosts and many animal
species are the intermediate hosts. Among these intermediate
hosts, feral pigs are infected with T .gondii have been found
to be infected at diferent rates between 4.4 to 45.9% (1).
Antibodies to T. gondii have been reported in wild boars
from Austria, Brazil, Czech Republic, France, Germany, Italy,
Japan, Slovak Republic, Spain, United States (Georgia and
South Carolina) (1, 2). Te meat of wild boars with tissue
cysts of T. gondii may be an important source for human
toxoplasmosis when people consume these meats under-
cooked or without control (3).
Neospora caninum, an important parasitic disease of
cattle, causes reproductive failure, especially abortion (4).
Neosporosis is primarily a disease of cattle and dogs but
antibodies have been detected a series of species, including
domestic, wildlife and zoo animals (5). Dogs can acquire
infection by ingestion of infected tissues; and intermedi-
ate hosts can be infected either by horizontal, postnatal or
by vertical transmission (6). Due to the economic impact
of neosporosis, existing studies are concentrated on farm
animals and there is limited serological data about wild life
(4-6). Anti-N. caninum antibodies have been reported in wild
boars from Spain and the Czech Republic (7, 8).
For both T. gondii and N. caninum wild pigs may be
important indicators for following environmental contamina-
Detection of Toxoplasma gondii and Neospora caninum antibodies in
Wild Boars (Sus scrofa) in Eastern Turkey
Balkaya, I.,
1
* Utuk, A. E.,
2
Babur, C.,
3
Beyhan, Y. E.,
4
Piskin, F. C.
5
and Sozdutmaz, I.
6
1
Ataturk University, Faculty of Veterinary Medicine, Department of Parasitology, Erzurum, Turkey.
2
Cukurova University, Faculty of Veterinary Medicine, Department of Parasitology, Adana, Turkey.
3
Refk Saydam National Hygiene Center, Communicable Diseases Research Department, Parasitology Laboratory, Ankara, Turkey.
4
Yuzuncu Yil University, Faculty of Medicine, Department of Medical Parasitology, Van, Turkey.
5
Veterinary Control Central Research Institute, Parasitology and Bee Diseases Laboratory, Ankara, Turkey.
6
Erciyes University, Faculty of Veterinary Medicine, Department of Virology, Kayseri, Turkey.
*
Corresponding Author: Ibrahim Balkaya, Address: AtaturkUniversity, Faculty of Veterinary Medicine, Department of Parasitology, Erzurum, Turkey.
Tel: +90-0442-2315532. Email: balkayaibrahim@hotmail.com
ABSTRACT
Te aim of this study was to examine for the presence of anti-Toxoplasma gondii and Neospora caninum
antibodies in wild boars and to study the impact of infection between the sylvatic and domestic life cycles
of these apicomplexan parasites. For this purpose, sera were collected from hunter-killed wild boars (Sus
scrofa) during the winter period of 2011 from Erzurum province of Turkey. Collected sera were examined for
antibodies against T. gondii and N. caninum by Sabin Feldman dye test (SFDT) and competitive-enzyme-
linked immunosorbent assay (c-ELISA), respectively. Out of 12 collected samples, 4 (33.3%) of sera were
found to be seropositive at the dilution of 1:16 for T. gondii however no seropositivity in any of the samples
was detected against N. caninum. To the best knowledge of the authors, this is the frst serologic study to
detect anti-T. gondii antibodies in wild boars in Turkey.
Keywords: Wild Boar; Sus scrofa; Eastern Turkey; Toxoplasma gondii; Neospora caninum;
Sabin-Feldman; c-ELISA.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 29 Toxoplasma gondii in Wild Boars in Turkey
tion since they are omnivorous animals obtaining infection
from their local environment (9). Serologic tests (IFA, MAT,
LAT, DT and c-ELISA) are used to monitor anti-T. gondii
and N. caninum antibodies in wild pigs (1, 8, 9).
Te aim of this study was to investigate the presence of
anti-T. gondii and N. caninum antibodies in wild boars and to
examine the impact between the sylvatic and domestic life of
pigs in relation to these apicomplexan parasites.
MATERIAL AND METHODS
Sample collection
Blood samples were collected from 12 wild boars that were
provided by certifed hunters from Horasan and Pasinler
regions of Erzurum province in Turkey in the year 2011.
From information provided by hunters, the majority of these
wild boars were adult in the age range 1-3 years, and all of
them were female. Te blood samples were collected for
serological studies from the heart, following the death of
animals after shooting. Te blood samples were transferred
into vacuum tubes, allowed to clot and then centrifuged at
4000 rpm for 10 minutes at room temperature. Subsequently
they were placed into eppendorf tubes and stored at -20°C
until use.
Serologic examination
All sera were examined for T. gondii antibodies using the
SF dye test (SFDT) as described (10). Te examinations
were carried out at the National Reference Laboratory for
Parasitology, Public Health Institution of Turkey. Te pro-
cedure included two steps in preparation for performing the
test. Healthy 3-4 week old white Swiss albino mice were
injected with the virulent RH strain of T. gondii. T. gondii RH
antigen was collected from the peritoneal fuid of mice after
48 hrs post injection. As an activator serum, human serum
seronegative for T. gondii was used including factors such as
magnesium, properdin, C
2
, C
3
, C
4
. Alkaline methylene blue
dye was prepared with 9.73 ml of 0.53 % Na
2
CO
3
(Sigma,
Seelze, Germany), 0.27 ml of 1.91 % Na
2
B4O
7
.10H
2
O
(Merck, NJ, USA) and 25 mg of methylene blue (Difco,
Detroit, MI, USA). Following inactivation of complement
at 56°C for 30 minutes, 25 µl of test sera were prepared
with normal saline in dilutions of 1:4, 1:16, 1:64, 1:256 and
1:1024. Te antigen was added to the sera preparations of
25 µl activator serum at approximately 25 T. gondii tachyzo-
ites observed in a microscopic feld of 40X magnifcation,.
Te mixture was incubated in a water-bath at 37°C for 50
minutes. 25 µl of alkaline methylene blue was added to the
mixture and kept in 4°C for 10 minutes. Examination was
carried out using light microscopy with 40X objective to
gauge whether T. gondii tachyzoite were stained. If more
than 50 % of tachyzoites on one microscopic feld were not
stained, this dilution step was accepted as positive. Titers of
1:16 and greater were considered as positive (11). Positive and
negative controls, which were confrmed by IFAT method,
were included in the above procedure.
Serologic examination (c-ELISA)
Antibodies to N. caninum were detected by using a commer-
cially available competitive enzyme-linked immunosorbent
assay (c-ELISA) kit (VMRD, USA). Te test was done by
following the instructions of manufacturer. Te mean optical
density (OD) at 630 nm was determined for all wells using
a microplate reader (ELx 800 UV, Universal Microplate
Reader, Bio-Tek Instruments, Invc., Winooski, VT, USA).
Te percent inhibition for each test sample was determined
using the below mentioned formula:
Inhibition (%) = 100 - [( Sample O.D. X 100)
÷ (Mean Negative Control O.D.)]
Te samples with values of ≥ 30% inhibition were re-
garded as positive and those with the values < 30% inhibition
were regarded as negative (12).
RESULTS
Anti-T. gondii antibodies in SFDT test were detected in 4 of
12 (33.3%) wild boar sera to be seropositive at 1:16 dilution
and, no seropositive cases (0%) were detected for N. caninum
antibodies.
DISCUSSION
Te wild boar is the only member of the suidae family present
in Turkey and bioecological data about this animal are limited
(13). Except in touristic regions, pork meat consumption is
restricted in Turkey; however, wild boars are important game
species and have the potential to cause damage to agricultural
crops in Turkey (13). When the wild boar population in-
creases, hunting organizations are arranged to cull wild boars
and in so doing decrease the crop damage. Due to restricted
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Balkaya, I. 30
meat consumption, hunted animals are often left behind,
close to the farm lands and consumed by domestic and wild
carnivores. Tis scenario leads wild boars into importance
in the sylvatic and domestic life cycles of T. gondii and N.
caninum.
Studies concerning N. caninum infections in wild boars
is limited (8, 14). Almería et al. studied 298 wild boars in
Spain and found a prevalence of 0.3% (14). Bártova et al.
studied on 565 wild boars and they detected the prevalence
as 18.1% in Czech Republic (8). Researchers used c-ELISA
for monitoring and IFAT for confrmation. In our study we
could not detect any seropositivity with c-ELISA. In do-
mestic pigs, experimental and natural infections have shown
a low N. caninum incidence that suggests that environmental
exposure to N. caninum is rare. Tis opinion may explain why
we could not detect seropositivity for wild boars in our survey
(14). Terefore, it appears that wild boar may not play an
important role between the sylvatic and domestic life cycle
of N. caninum infection.
Ranucci et al. studied 400 wild boars in Italy and found
the prevalence of T. gondii as 14% using IFAT (2). Gauss et
al. (15) studied on 507 wild boars and found the prevalence
of T. gondii 38.4% with MAT in Spain. Richommeet et al.
studied 148 wild boars and found the prevalence 17.6% with
MAT in France (16). Hejlícek et al. (17) studied on 124 wild
boars and found a prevalence of 15% with SFDT in the
Czech Republic. Furthermore the prevalence rates of T.gondii
were found as 18.2% (n=170) and 34.4% (n=257) in Georgia
and South Carolina provinces of United States respectively
with MAT, 4.4% (n=90) and 5.6% (n=108) in Kukamoto and
Iriomote Island of Japan respectively with LAT (1).
In our study we detected T. gondii antibodies in 4 out of
12 (33.3%) animals with SFDT. Te diferences between the
results may be associated with diferent climatic conditions,
sample sizes, serological tests used and diferent species of
fnal hosts, their population density and their feeding behav-
iors. Although our sample size is small, 33.3% prevalence may
be important for the transition between sylvatic to domestic
life cycles by domestic and wild felids.
Limitations of this study include the small sample size
and the fact that only female wild pigs were tested. In this
regard this study only gives an indication of the distribution
of these diseases among wild pigs may be considered a pre-
liminary study to further larger and wider ranging studies in
this area of Turkey. Diferences in infection rates between the
sexes has not been extensively studied for either Toxoplasma
gondii and Neospora caninum although a study in dogs has
demonstrated a greater prevalence of N. caninum antibodies
in female dogs than in males (18, 19).
In conclusion, it is hoped that the results of this pre-
liminary study will contribute to the understanding of these
apicomplexan parasites’ epidemiology in the wild life. Further
studies are required for developing efective control programs
and a clear understanding of these diseases both in Turkey
and worldwide.
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