- Email: [email protected]
Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia
Accepted Manuscript Title: Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia ˇ Author: Marija Markovi´c Vladimir Ivovi´c Tijana Stajner Vitomir Djoki´c Ivana Klun Branko Bobi´c Aleksandra Nikoli´c Olgica Djurkovi´c-Djakovi´c PII: DOI: Reference:
S0147-9571(14)00018-6 http://dx.doi.org/doi:10.1016/j.cimid.2014.03.001 CIMID 961
To appear in: Received date: Revised date: Accepted date:
5-11-2013 27-2-2014 3-3-2014
ˇ Please cite this article as: Markovi´c M, Ivovi´c V, Stajner T, Djoki´c V, Klun I, Bobi´c B, Nikoli´c A, Djurkovi´c-Djakovi´c O, Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia, Comparative Immunology, Microbiology and Infectious Diseases (2014), http://dx.doi.org/10.1016/j.cimid.2014.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ORIGINAL ARTICLE
2 3 4
6
ip t
5
Evidence for genetic diversity of Toxoplasma gondii in selected
8
intermediate hosts in Serbia
9
us
10
cr
7
11
Marija Marković, Vladimir Ivović, Tijana Štajner, Vitomir Djokić, Ivana Klun,
13
Branko Bobić, Aleksandra Nikolić, Olgica Djurković-Djaković*
an
12
14
National Reference Laboratory for Toxoplasmosis, Institute for Medical Research,
16
University of Belgrade, Dr. Subotića 4, P.O. Box 102, 11129 Belgrade, Serbia
M
15
20 21 22 23 24 25 26 27
te
19
Running title: Toxoplasma genotypes in Serbia
Ac ce p
18
d
17
28 29 30 31 32
* Corresponding author. Tel.: +381 11 2685 788; fax: +381 11 2643 691. E-mail address: [email protected] (O. Djurković-Djaković).
33 1
Page 1 of 26
34
ABSTRACT
36
To contribute to the insight into the worldwide population structure of Toxoplasma gondii, we
37
genetically characterized a total of eight strains isolated from intermediate hosts including
38
humans, sheep and pigeons in Serbia. Although parasite DNA was detected in 28.2% (60/213)
39
of the human samples from 162 patients serologically suspected of active toxoplasmosis, as
40
well as in 5/7 seropositive pigeons and in 2/12 seropositive sheep examined, multilocus PCR-
41
RFLP genotyping, using SAG1, 5’SAG2, 3’SAG2, GRA6, 5’GRA7 and 3’GRA7 as markers,
42
was successful in only four human isolates (of which one was isolated from both the
43
bronchoalveolar lavage fluid and blood samples of a single patient), one sheep and three
44
pigeons. Of the eight isolates, five were type II (62.5%), one was type III, one was atypical,
45
and one had a type I allele at GRA6 as the single locus genotyped. Although type II, as
46
elsewhere in Europe, predominated, these results may suggest a higher genetic diversity of T.
47
gondii in Serbia, reflecting local environmental contamination and also the geographical
48
position of the country in South-East Europe. deletion
cr
us
an
M
d
te
Ac ce p
49 50
ip t
35
51
Key words: Toxoplasma gondii, intermediate hosts, humans, sheep, pigeons, genotyping,
52
PCR-RFLP, Serbia, South-East Europe
53 54 55 56 57
2
Page 2 of 26
57
Introduction Toxoplasma gondii, a ubiquitous protozoan, infects a broad range of mammals and birds
59
which all act as intermediate hosts, whereas felids are the only definitive hosts. In
60
intermediate hosts, following a brief acute stage characterized by the presence of circulating
61
tachyzoites, parasites convert to tissue cysts mainly localized in the muscular and neural
62
tissues. In felids, sexual reproduction of the parasite results in the production of oocysts shed
63
with feces, thereby contaminating the environment. Infection with either parasite form is
64
mainly peroral, and consumption of meat containing tissue cysts and ingestion of oocysts
65
from water and soil (through gardening and farming) have been shown to be major routes of
66
human infection [1,2]. Toxoplasmosis in humans is generally benign, but may have serious
67
consequences in the developing fetus in case of maternal infection in pregnancy and in
68
immunosuppressed individuals. In animals, T. gondii infection can cause various health
69
problems, ranging from mild to serious infectious disease symptoms to reproductive issues,
70
and even disseminated infection and death deletion [3]. The risk to human and animal health,
71
in line with the One Health concept, warrants evaluation of T. gondii distribution in the
72
environment. As the detection of T. gondii oocysts in water and soil is cumbersome and not
73
yet feasible on a large scale, assessing the presence of the parasite in its herbivore
74
intermediate hosts is a good indicator of environmental contamination.
Ac ce p
te
d
M
an
us
cr
ip t
58
75
The population structure of T. gondii is unexpectedly oligoclonal for a parasite whose life
76
cycle involves sexual reproduction. Initially, it was characterized by three main clonal
77
lineages designated as type I, II and III that were found, with the predominance of type II, in
78
Europe and North America [4-6]. However, as data for South America and Africa became
79
available, a higher frequency of non-clonal strains (atypical and recombinant) was revealed,
80
indicating that the population structure is more diverse than previously thought [7-10].
81
Moreover, specific African non-clonal genotypes, termed Africa 1, 2 and 3, have been 3
Page 3 of 26
82
identified [11, 12]. But the picture is currently being complicated in the Western World as
83
well, since a fourth clonal lineage has been recently described in North America [13]. Strain genotype has been associated with clinical severity of human toxoplasmosis [4].
85
Type II strains have been shown to be most prevalent in congenital infection and AIDS
86
patients in North America and Europe [5,6,14]. Atypical strains have been associated with
87
severe toxoplasmosis in immunocompetent patients [15,16], particularly in South America
88
where they correlate with ocular toxoplasmosis [7], and in the setting of immunosuppression
89
[17]. In animal hosts, emerging information indicates severe histopathological lesions in
90
sheep abortions caused by T. gondii of an atypical genotype [18], but also the association of
91
type II strains with fatal toxoplasmosis in a cat [19], arctic foxes [20] and wild hares [21].
an
us
cr
ip t
84
Information on the population structure of T. gondii is important in view of the possible
93
health implications. However, data are quite scarce for South-East Europe, Serbia included,
94
where the genotype of a single human isolate has been reported [22] and virtually none in
95
animals. The aim of this study was thus to identify T. gondii genotypes circulating in Serbia
96
by detecting the parasite in three of its intermediate hosts.
97
1. Materials and Methods
98
2.1. Study area
Ac ce p
te
d
M
92
99
Located in South-East Europe, Serbia (without Kosovo) extends over a territory of 77,512
100
km2 and has a population of 7.5 million. Belgrade with its surroundings (Belgrade District) is
101
a highly urban region, with a population of approximately 1.6 million (Fig. 1A).
102
2.2. Collection of samples
103
Genotyping was attempted from both human and animal biological materials.
4
Page 4 of 26
Human materials involved a total of 213 body fluid samples (Table 1) from 162 patients
105
both clinically and serologically suspected of active toxoplasmosis, defined as infection stages
106
characterized by the presence of tachyzoites in body fluids. These included adults suspected
107
of acute toxoplasmosis, women suspected of infection during pregnancy and neonates
108
suspected of congenital toxoplasmosis (CT), patients with ocular and cerebral toxoplasmosis
109
and patients who underwent hematopoietic stem cell transplantation (HSCT), who were
110
referred to the National Reference Laboratory for Toxoplasmosis (NRLToxo) between 2008
111
and 2012. Since NRLToxo is the single laboratory in Serbia to perform expert diagnosis,
112
patients originated from all over the country.
us
cr
ip t
104
Animals tested were sheep (Ovis aries) and pigeons (Columba livia) and materials
114
included blood samples and hearts. Sheep samples from a total of 15 ewes (mean age 14.7
115
months, range 13-24 months), were collected from two abattoirs in the vicinity of Belgrade
116
(in the administrative municipalities of Pećinci and Stara Pazova), which serve the local farms
117
(Fig. 1B). All sheep were destined for human consumption. Pigeons were collected within the
118
scope of an ongoing large ecological investigation of feral pigeons as indicators of
119
environmental contamination led by the Natural History Museum in Belgrade. For this study,
120
hearts and blood samples from 72 pigeons captured at 15 collection sites, mainly in downtown
121
Belgrade (Fig. 1B) were made available to us.
122
2.3. Serology
Ac ce p
te
d
M
an
113
123
Blood samples were initially examined for T. gondii specific antibodies. Human samples
124
were tested for specific IgG and IgM antibodies and avidity of specific IgG antibodies using
125
commercial tests on a VIDAS analyzer (bioMérieux, Marcy l’Etoile, France). Positivity
126
thresholds were 8 IU/mL for IgG and 0.65 for IgM, while avidity was expressed in index
127
values (low: I < 0.2; intermediate: 0.2 ≤ I < 0.3; high: I ≥ 0.3). In some cases, specific IgM 5
Page 5 of 26
antibodies were additionally tested by the immunosorbent agglutination assay (ISAgA,
129
bioMérieux, index scale 0-12, positivity threshold I = 9). Commercial assays were performed
130
according to the manufacturer’s instructions. Specific IgG antibodies were also tested by an
131
in-house high sensitivity agglutination test (cut-off value = 1:20) [23]. A slight adaptation of
132
this test (the modified agglutination test, MAT) was used for animal samples [24]. Sheep and
133
pigeon sera were serially two-fold diluted starting at 1:10 and sera reactive at ≥ 1:10 were
134
considered positive [25]. In bioassay experiments, the cut-off was set at 1:20.
135
2.4. Study design
us
cr
ip t
128
In human biological materials obtained from patients serologically suspected of active
137
toxoplasmosis, genotyping was attempted both directly and, in an attempt to isolate and
138
propagate the parasite, following a bioassay step. In animal samples, DNA was extracted from
139
the hearts of seropositive sheep and pigeons following trypsin digestion, and PCR-positive
140
hearts were further bioassayed in mice.
M
d
The study followed the tenets of the Declaration of Helsinki and was approved by a local
te
141
an
136
(Institute for Medical Research) Ethics Committee.
143
2.5. Trypsin digestion of animal tissue
Ac ce p
142
144
Whole animal hearts (sheep ! 200g, pigeon 5-6 g) were mixed in an electric blender and
145
incubated at 37 °C for 1.5 h with trypsin (final concentration 0.25%) and antibiotics [peni-
146
strepto (PAA Laboratories GmbH, Pasching, Austria) and amoxicillin (Hemofarm, Vršac,
147
Serbia)]. The suspension was then filtered and washed with saline three times. The obtained
148
pellet was resuspended in saline, to 2 mL for sheep and to 1 mL for pigeon material. Part (200
149
! L) of the homogenated tissue was analyzed by PCR and the remaining tissue of the positive
150
samples was bioassayed.
151
2.6. Bioassay 6
Page 6 of 26
Bioassay was performed by intraperitoneal inoculation of suspected material (! 500 ! L),
153
with addition of gentamycin (Galenika, Zemun, Serbia), into two Swiss-Webster female mice
154
per sample, as previously described [26]. After six weeks the mice were euthanized, blood
155
was tested by MAT and brains were homogenized with 1 mL of saline each for microscopic
156
examination for T. gondii cysts. Cysts were enumerated at a sensitivity threshold of 10 cysts
157
per brain. A bioassay was considered positive in case of positive serology, detection of brain
158
cysts, or both, and DNA was extracted from the brains of all positive mice.
159
2.7. DNA extraction
us
cr
ip t
152
DNA was extracted from 200 ! l of biological materials using the QIAmp DNA mini kit
161
(Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Extracted
162
DNA was resuspended in 150 ! l of nuclease-free water and stored at -20°C.
163
2.8. Detection of T. gondii DNA by Real-Time PCR
d
M
an
160
T. gondii DNA was detected by Real-Time PCR (RT-PCR) targeted at the 529 bp
165
repetitive element (gene bank accession number AF146527), as previously described [27].
166
Briefly, the PCR reaction was performed in a final volume of 20 μL mixture containing 10 μl
167
Maxima™ Probe/ROX qPCR Master Mix (2X) [Fermentas (Thermo Fisher Scientific),
168
Waltham, MA, USA], 0.25 mM of each primer, 0.10 mM of TaqMan probe (Invitrogen, Life
169
Technologies, Carlsbad, CA, USA), 0.015 U/μL of UNG, 25 mM MgCl2 and nuclease-free
170
water, plus 3 μL of extracted DNA. Amplification was performed over 40 cycles: 2 min at 50
171
ºC for UNG pre-treatment, 10 min at 94 ºC initial denaturation, followed by 40 cycles of 15 s
172
at 95 ºC for denaturation and 60 s at 60 ºC for annealing/extension.
173
2.9. Genotype analysis of T. gondii by multilocus PCR-RFLP
Ac ce p
te
164
7
Page 7 of 26
Genotyping was performed by the PCR-restriction fragment length polymorphism (PCR-
175
RFLP) method using six markers including SAG1, 5’SAG2, 3’SAG2, GRA6, 5’GRA7 and
176
3’GRA7, across four genetic loci (Table 2). For each marker the PCR reaction mixture
177
consisted of 12.5 μL PCR Master Mix (2X) (Fermentas), 1 μL 10 μМ forward and reverse
178
primer, 7.5 μL nuclease free water and 3 μL DNA extracted from the sample in a 25 μL
179
reaction volume. For each marker the PCR program was different (Table 3). Positive controls
180
consisted of T. gondii type I (RH), type II (Me49) and type III (NED) strains, while nuclease
181
free water was used as a negative control. To confirm DNA amplification, PCR products were
182
separated by electrophoresis in 3% agarose gel, and digested with appropriate restriction
183
enzymes for different markers. The PCR mixture for digestion consisted of 12 μL of nuclease-
184
free water, 2.5 μL of buffer, 0.5 μL of restriction enzyme (concentration 5 U/µL for MbO II,
185
10 U/µL for all others), and 10 μL of PCR product. RFLPs were visualized by electrophoresis
186
in 3% agarose gel stained with ethidium bromide. Estimation of fragment size was based on
187
comparison to a 50 bp DNA ladder (Fermentas).
188
2. Results
Ac ce p
te
d
M
an
us
cr
ip t
174
189
A total of 213 biological samples from 162 patients serologically suspected of active
190
toxoplasmosis was tested by RT-PCR. T. gondii DNA was detected in 60 (28.2%) samples
191
from 52 individuals (Table 1). Of the 132 bioassay experiments, 30 were positive for cysts
192
and/or serologically.
193
T. gondii-specific antibodies were found in all tested sheep. DNA was extracted from the
194
hearts of 12 animals, of which two were RT-PCR positive. Due to technical reasons, only one
195
was bioassayed, leading to the isolation of a T. gondii strain (Table 4).
196
Seven of the 72 pigeons (9.7%, 95% CI 2.9-16.5) were seropositive for T. gondii and DNA
197
was extracted from the hearts of all seropositive animals. T. gondii DNA was detected in five 8
Page 8 of 26
198
animals, of which four were bioassayed and all four bioassays were positive (three based on
199
the detection of cysts of which one at an extremely high cyst burden of ~2000 cysts per brain;
200
one only by MAT) (Table 4). Based on previous experience in our laboratory that genotyping is feasible only in samples
202
which are RT-PCR positive at a cycle threshold (Ct) value of below 30, it was attempted in a
203
total of 19 human samples. However, as many of those were actually PCR-positive at a Ct
204
close to 30, and the harvest of brain cysts was poor in many bioassay experiments, genotypes
205
of four human isolates were determined. Of these, two belonged to type II, one was non-
206
clonal and one, typed only on GRA6, had a type I allele (Table 5). The non-clonal strain,
207
isolated from the blood and BAL fluid of a single patient, was type II according to SAG1,
208
5’SAG2, 3’SAG2 and GRA6, but had alleles of both type I and II on 5’GRA7 and 3’GRA7,
209
indicating that it could be atypical, which was later confirmed by microsatellite analysis [17].
210
This strain was also the single one directly typed from both the blood and BAL samples.
d
M
an
us
cr
ip t
201
Genotyping of animal isolates was attempted from strains isolated by bioassay from four
212
pigeons and one sheep and was successful in three pigeons and one sheep; of the three T.
213
gondii isolates from pigeons two were type II and one was type III, while the strain isolated
214
from sheep belonged to the type II lineage (Table 5).
215
3. Discussion
Ac ce p
te
211
216
Genotyping of T. gondii isolates in Serbia revealed evidence of the presence of several
217
genetic types. Of the eight T. gondii strains isolated from three intermediate host species
218
(humans, sheep and pigeons), type II was predominant (62.5%, 5/8), but one (12.5%)
219
belonged to type III, one was atypical, and one, only genotyped at the GRA6 locus, had a type
220
I allele. The latter finding, however, should be taken with caution; using only GRA6 which
221
may distinguish among clonal lineages was at one time considered to be sufficient for 9
Page 9 of 26
222
European strains where only these are expected [28], typing of a single allele does not
223
necessarily imply that all alleles are of the same type [29]. Isolation of these different genotypes among eight isolates in a rather small territory may
225
suggest a greater genetic diversity than would be expected from other studies in Europe. In
226
humans, studies in France showed a large predominance of type II isolates (>80%), with the
227
occurrence of some type I and type III isolates [6,14]. Few studies of human isolates have
228
been performed elsewhere in Europe. Nowakowska et al. [30] reported that among cases of
229
CT in Poland, all nine genotyped isolates were type II. Two human strains isolated from CT
230
cases in Serbia and recently in the neighboring Romania, both belonged to type II [22,31].
us
cr
ip t
224
Type II is also vastly predominant in animals in Europe, such as in chicken in Austria [32],
232
in cats in Germany [33], and even in wildlife such as carnivorous mammals [34], rodents and
233
foxes [35], arctic foxes [20] and hares [21]. In this study, we analyzed sheep and pigeons as
234
species that may be considered markers of environmental contamination. Sheep are
235
herbivores and as such get infected mainly by ingesting T. gondii oocysts by grazing on
236
pastures or through contaminated water or feed. All 15 sheep analyzed in this study were
237
previously exposed to T. gondii as shown by 100% seropositivity, and the single genotyped
238
isolate was characterized as type II. This is in agreement with previous work on sheep. In a
239
study in England where isolation of T. gondii was performed from ovine abortion tissue, all
240
13 isolated strains belonged to type II [36]. In France, analysis of eight and 46 strains isolated
241
from ovine meat destined for human consumption, showed that all were type II except a
242
single one which belonged to type III [37,38].
Ac ce p
te
d
M
an
231
243
Pigeons are free range animals that also get infected feeding from the ground. Pigeons live
244
in close association with humans and their daily migrations are relatively limited. It has been
245
shown that, in search for food, they rarely cross distances over 0.5-0.6 km [39], reflecting
246
environmental contamination of the particular area where they are captured. And as prey to 10
Page 10 of 26
cats, pigeons may also contribute to environmental dissemination of T. gondii. Few studies
248
have analyzed T. gondii genotypes of isolates from birds in general; of the 12 genotyped
249
pigeon isolates in Portugal, Waap et al. [40] showed that nine were type II, but two type III
250
and one type I were isolated as well. We here report that among the three genotyped pigeon
251
isolates in Serbia, two were type II while one was type III. On the other hand, predominance
252
of type III over type II isolates was shown in several bird species in Egypt and Iran [41,42].
cr
ip t
247
The described vast predominance of type II among both human and animal isolates
254
elsewhere in Europe does not seem to apply to the Mediterranean, where type III in particular
255
was much more frequent, detected in as many as 80% of the human and rodent isolates in the
256
islands of Cyprus and Crete [43]; and in 33%, 26% and 16%, respectively, of the strains
257
isolated from chickens, pigs and pigeons in Portugal [44,45,40]. In Spain, out of 25 human
258
isolates, type I, II and III have been detected at a ratio of 40, 40, and 20%, respectively [46].
259
Since these studies (with the exception of Waap et al. [40] who used five microsatellite
260
markers) were based on the detection of a single marker, i.e. GRA6 or SAG2, which although
261
capable of differentiating among the three clonal types, can be insufficient to detect and
262
resolve atypical alleles from type III and to a lesser extent, from type I [28,47,48], it is
263
possible that there may have been atypical strains among those reported as type III; if so, the
264
Mediterranean area may be characterized by a larger genetic diversity compared to
265
continental Europe.
Ac ce p
te
d
M
an
us
253
266
Since Serbia borders with the Mediterranean area, it may not be surprising that, as
267
presented here, intermediate hosts harbored genetically diverse strains. Use of additional
268
genetic markers to the six ones we used may possibly identify other alleles, characterizing
269
non-clonal strains, thereby even increasing the observed diversity. The presented T. gondii
270
genetic diversity may be all the more important since it has been observed in a relatively
271
small geographical area. The findings plausibly reflect the geographical position of Serbia in 11
Page 11 of 26
South-East Europe, in the centre of the Balkan Peninsula, an intersection between Europe and
273
Asia, and in relative proximity to Africa. Similarly, the recent finding of the Africa 1
274
genotype in two human isolates from Turkey was associated with the geographical position of
275
Turkey between Asia, Europe and Africa [49]. A higher genetic diversity including the
276
presence of non-clonal T. gondii in Serbia may indicate phylogenetic ties among Asian,
277
African and European T. gondii populations, or, in evolutionary terms more recent
278
environmental contamination which may occur from migratory birds that often stopover in
279
South-East Europe during their seasonal movement from Africa to the North. A relatively
280
novel factor that may contribute to T. gondii genetic diversity involves globalization of food
281
including importation of meats from areas of a highly divergent population structure.
282
Irrespective of the origin, the public health and veterinary implications of the genetic diversity
283
of T. gondii warrant more research in this and other areas of the world to fully appreciate the
284
organism’s population structure and its ramifications.
285
287
288 289 290
Conflict of interests
The authors declare no conflict of interests.
Ac ce p
286
te
d
M
an
us
cr
ip t
272
Acknowledgements
The results of this paper have been presented in part at the 11th European Multicolloquium of Parasitology (EMOP XI), Cluj-Napoca, Romania, 25-29 July, 2012. deletion
291
The authors are grateful to Isabelle Villena (Reims, France) for kindly supplying the
292
antigen for HSDA, and the reference DNA for type III (NED strain). We also wish to
293
acknowledge Marko Raković, Natural History Museum of Belgrade, for kindly letting us the
294
pigeon samples.
12
Page 12 of 26
This study was supported by a grant (project No. III 41019) from the Ministry of
295
Education, Science and Technological Development of Serbia.
297
References
298
[1]
ip t
296
Bobić B, Jevremović I, Marinković J, Šibalić D, Djurković-Djaković O. Risk factors for Toxoplasma infection in a reproductive age female population in the area of Belgrade,
300
Yugoslavia. Eur J Epidemiol 1998;14:605-10. [2]
Cook AJC, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, et al. on behalf
us
301
cr
299
of the European Research Network on Congenital Toxoplasmosis. Sources of
303
toxoplasma infection in pregnant women: European multicentre casecontrol study. Brit
304
Med J 2000;321:142-7.
an
302
[3]
Dubey JP. Toxoplasmosis of animals and humans. CRC Press, Boca Raton, FL; 2010.
306
[4]
Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation
1997;35:1411-4.
310
[6]
Ajzenberg D, Cogné N, Paris L, Bessières MH, Thulliez P, Filisetti D, et al. Genotype
of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and
312
correlation with clinical findings. J Infect Dis 2002;186:684-9.
313 314
Ac ce p
Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol
309
311
Howe DK, Honore S, Derouin F, Sibley LD. Determination of genotypes of
te
[5]
d
of parasite genotype with human disease. J Infect Dis 1995;172:1561-6.
307 308
M
305
[7]
Khan A, Jordan C, Muccioli C,Vallochi AL, Rizzo LV, Belfort Jr R, et al. Genetic
315
divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil.
316
Emerg Infect Dis 2006;12:942-9.
13
Page 13 of 26
317
[8]
Al-Kappany YM, Rajendran C, Abu-Elwafa SA, Hilali M, Su C, Dubey JP. Genetic
318
diversity of Toxoplasma gondii isolates in Egyptian feral cats reveals new genotypes. J
319
Parasitol 2010;96:1112-4. [9]
Ferreira IM, Vidal JE, de Mattos Cde C, de Mattos LC, Qu D, Su C, Pereira-Chioccola
ip t
320
VL. Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human
322
patients in Sao Paulo State, Brazil identified distinct genotypes. Exp Parasitol
323
2011;129:190-5.
cr
321
[10] Demar M, Hommel D, Djossou F, Peneau C, Boukhari R, Louvel D, et al. Acute
325
toxoplasmosis in immunocompetent patients hospitalized in an intensive care unit in
326
French Guiana. Clin Microbiol Infect 2012;18:221-31.
an
us
324
[11] Ajzenberg D, Bañuls AL, Su C, Dumètre A, Demar M, Carme B, Dardé ML. Genetic
328
diversity, clonality and sexuality in Toxoplasma gondii. International J Parasitol
329
2004;34: 1185-96.
d
M
327
[12] Mercier A, Devillar S, Ngoubangoye B, Bonnabau H, Bañuls AL, Durand P, et al.
331
Additional haplogroups of Toxoplasma gondii out of Africa: population structure and
332
mouse-virulence of strains from Gabon. PLOS Negl Trop Dis 2010;4,876.
Ac ce p
te
330
333
[13] Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD. Genetic analyses of
334
atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Int J
335
Parasitol 2011;41:645-55.
336
[14] Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, et al. Genotype of 88
337
Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised
338
patients and correlation with clinical findings. J Infect Dis 2009;199:1155-67.
339
[15] Carme B, Bissuel F, Ajzenberg D, Bouyne R, Aznar C, Demar M, et al. Severe acquired
340
toxoplasmosis in immunocompetent adult patients in French Guiana. J Clin Microbiol
341
2002;40:4037-44. 14
Page 14 of 26
342
[16] Delhaes L, Ajzenberg D, Sicot B, Bourgeot P, Dardé ML, Dei-Cas E, Houfflin-Debarge
343
V. Severe congenital toxoplasmosis due to a Toxoplasma gondii strain with an atypical
344
genotype: case report and review. Prenat Diagn 2010;30:902-5. [17] Štajner T, Vasiljević Z, Vujić D., Marković M, Ristić G, Mićić D, et al. Atypical strain
346
of Toxoplasma gondii causing fatal reactivation after haematopoietic stem cell
347
transplantion in a patient with an underlying immunological deficiency. J Clin
348
Microbiol 2013; 51, 2686-90.
cr
ip t
345
[18] Edwards JF, Dubey JP. Toxoplasma gondii abortion storm in sheep on a Texas farm and
350
isolation of mouse virulent atypical genotype T. gondii from an aborted lamb from a
351
chronically infected ewe.Vet Parasitol. 2013;192,129-36.
an
us
349
[19] Spycher A, Geigy C, Howard J, Posthaus H, Gendron K, Gottstein B, et al. Isolation
353
and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an
354
immunocompetent 10-year-old cat. J Vet Diagn Invest. 2011;23,104-8.
d
M
352
[20] Prestrud KW, Asbakk K, Mørk T, Fuglei E, Tryland M, Su C. Direct high-resolution
356
genotyping of Toxoplasma gondii in arctic foxes (Vulpes lagopus) in the remote arctic
357
Svalbard archipelago reveals widespread clonal Type II lineage.Vet Parasitol.
Ac ce p
358
te
355
2008;158:121-8.
359
[21] Jokelainen P, Isomursu M, Näreaho A, Oksanen A. Natural Toxoplasma gondii
360
infections in European brown hares and mountain hares in Finland: proportional
361 362
mortality rate, antibody prevalence, and genetic characterization. J Wildl Dis. 2011;47:154-63.
363
[22] Djurković-Djaković O, Klun I, Khan A, Nikolić A, Knezević-Ušaj S, Bobić B, Sibley
364
LD. A human origin type II strain of Toxoplasma gondii causing severe encephalitis in
365
mice. Microb Infect 2006;8:2206-12.
15
Page 15 of 26
366
[23] Desmonts G, Remington JS. Direct agglutination test for diagnosis of Toxoplasma
367
infection: method for increasing sensitivity and specificity. J Clin Microbiol
368
1980;11:562-8.
370
[24] Dubey JP, Desmonts G. Serological responses of equids fed Toxoplasma gondii ocysts.
ip t
369
Eq Vet J 1987;19:337-9.
[25] Dubey JP, Graham DH, Blackston CR, Lehmann T, Gennari SM, Ragozo AM, et al.
372
Biological and genetic characterization of Toxoplasma gondii isolates from chickens
373
(Gallus domesticus) from São Paulo, Brazil: unexpected findings. Int J Parasitol
374
2002;32:99-105.
us
cr
371
[26] Djurković-Djaković O, Nikolić A, Bobić B, Klun I, Aleksić A. Stage conversion of
376
Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine
377
dithiocarbamate. Microb Infect 2005;7:49-54.
M
an
375
[27] Vujanić M, Ivović V, Kataranovski M, Nikolić A, Bobić B, Klun I, et al.
379
Toxoplasmosis in naturally infected rodents in Belgrade, Serbia. Vector Borne Zoon Dis
380
2011;11:1209-11.
382 383 384
te
[28] Fazaeli A, Carter PE, Darde ML, Pennington TH. Molecular typing of Toxoplasma
Ac ce p
381
d
378
gondii strains by GRA6 gene sequence analysis. Int J Parasitol 2000;30:637-42.
[29] Ajzenberg D. Type I strains in human toxoplasmosis: myth or reality? Future Microbiol. 2010;5:841-3.
385
[30] Nowakowska D, Colon I, Remington JS, Grigg M, Golab E, Wilczynski J, Sibley LD.
386
Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological
387
testing of samples from infants in Poland diagnosed with congenital toxoplasmosis. J
388
Clin Microbiol 2006;44:1382-9.
16
Page 16 of 26
389
[31] Costache CA, Colosi HA, Blaga L, Györke A, Pastiu AI, Colosi IA, Ajzenberg D. First
390
isolation and genetic characterization of a Toxoplasma gondii strain from a symptomatic
391
human case of congenital toxoplasmosis in Romania. Parasite 2013;20:11. [32] Dubey JP, Edelhofer R, Marcet P, Vianna MC, Kwok OC, Lehmann T. Genetic and
393
biologic characteristics of Toxoplasma gondii infections in free-range chickens from
394
Austria. Vet Parasitol 2005;133:299-306.
cr
ip t
392
[33] Herrmann DC, Pantchev N, Globokar Vrhovec M, Barutzki D, Wilking H, Fröhlich A,
396
et al. Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in
397
Germany. Int J Parasitol 2010;40:285-92.
us
395
[34] Burrells A, Bartley PM, Zimmer IA, Roy S, Kitchener AC, Meredith A, et al.
399
Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian
400
carnivores
401
10.1017/S0031182013001169.
the
UK.
Parasitology
2013
[Epub
ahead
of
print].
doi:
d
in
M
an
398
[35] Herrmann DC, Maksimova P, Maksimova A, Sutora A, Schwarza S, Jaschkeb W, et al.
403
Toxoplasma gondii in foxes and rodents from the German Federal States of
404
Brandenburg and Saxony-Anhalt: Seroprevalence and genotypes. Vet Parasitol
406 407
Ac ce p
405
te
402
2012;185:78-85.
[36] Owen MR, Trees AJ. Genotyping of Toxoplasma gondii associated with abortion in sheep. J Parasitol 1999;85:382-4.
408
[37] Dumetre A, Ajzenberg D, Rozette L, Mercier A, Dardé ML. Toxoplasma gondii
409
infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping
410
by microsatellite analysis. Vet Parasitol 2006;142:376-9.
411
[38] Halos L, Thebault A, Aubert D, Thomas M, Perret C, Geers R, et al. An innovative
412
survey underlining the significant level of contamination by Toxoplasma gondii of
413
ovine meat consumed in France. Int J Parasitol 2010;40:193-200. 17
Page 17 of 26
414 415
[39] Rose E, Nagel P, Haag-Wackernagel D. Spatio-temporal use of the urban habitat by feral pigeons (Columba livia). Behav Ecol Sociobiol 2006;60:242-54. [40] Waap H, Vilares A, Rebelo E, Gomes S, Angelo H. Epidemiological and genetic
417
characterization of Toxoplasma gondii in urban pigeons from the area of Lisbon
418
(Portugal). Vet Parasitol 2008;157:306-9.
ip t
416
[41] Dubey JP, Graham DH, Dahl E, Hilali M, El-Ghaysh A, Sreekumar C, et al. Isolation
420
and molecular characterization of Toxoplasma gondii from chickens and ducks from
421
Egypt. Vet Parasitol 2003;114:89-95.
us
cr
419
[42] Khademvatan S, Saki J, Yousefi E, Abdizadeh R. Detection and genotyping of
423
Toxoplasma gondii strains isolated from birds in the southwest of Iran. Brit Poultry Sci
424
2013;54:76-80.
M
an
422
[43] Messaritakis I, Detsika M, Koliou M, Sifakis S, Antoniou M. Prevalent genotypes of
426
Toxoplasma gondii in pregnant women and patients from Crete and Cyprus. Amer J
427
Trop Med Hyg 2008;79:205-9.
te
d
425
[44] Dubey JP, Vianna MC, Sousa S, Canada N, Meireles S, Correia da Costa JM, et al.
429
Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal. J
430
Ac ce p
428
Parasitol 2006;92:184-6.
431
[45] de Sousa S, Ajzenberg D, Canada N, Freire L, da Costa JM, Darde ML, et al. Biologic
432
and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.
433
Vet Parasitol 2006;135:133-7.
434
[46] Fuentes I, Rubio JM, Ramirez C, Alvar J. Genotypic characterization of Toxoplasma
435
gondii strains associated with human toxoplasmosis in Spain: direct analysis from
436
clinical samples. J Clin Microbiol 2001;39:1566-70.
437 438
[47] Ajzenberg D, Dumètre A, Dardé ML. Multiplex PCR for typing strains of Toxoplasma gondii. J Clin Microbiol 2005;43:1940-3. 18
Page 18 of 26
439
[48] Su C, Zhang X, Dubey JP. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP
440
markers: a high resolution and simple method for identification of parasites. Int J
441
Parasitol 2006;36:841-8. [49] Döşkaya M, Caner A, Ajzenberg D, Değirmenci A, Dardé ML, Can H, et al. Isolation of
443
Toxoplasma gondii strains similar to Africa 1 genotype in Turkey. Parasitol Int
444
2013;62,471-4.
cr
ip t
442
[50] Richomme C, Aubert D, Gilot-Fromont E, Ajzenberg D, Mercier A, Ducrot C, et al.
446
Genetic characterization of Toxoplasma gondii from wild boar (Sus scrofa) in France.
447
Vet Parasitol. 2009;164:296-300.
us
445
Ac ce p
te
d
M
an
448
19
Page 19 of 26
448
LEGEND FOR FIGURE
449 450
FIG. 1. A: Map of Europe with Serbia shaded grey. B: Map of Serbia (Belgrade District in
452
black),
sheep collection site,
ip t
451
pigeon collection site; source: www.wikimedia.org.
Ac ce p
te
d
M
an
us
cr
453
20
Page 20 of 26
453
Peripheral blood Fetal blood Amniotic fluid Aqueous humor Cerebrospinal fluid BAL ∑
No. examined 138 14 38 12 10 1 213
% positive 24.6 21.4 28.9 50.0 50.0 100.0 28.2
te
d
M
an
BAL – bronchoalveolar lavage fluid
Ac ce p
456 457 458
RT-PCR No. positive 34 3 11 6 5 1 60
cr
Sample type
ip t
Table 1 Toxoplasma gondii DNA detection in human biological materials.
us
454 455
21
Page 21 of 26
458
Table 2
459
Genetic markers, primer sequences and restriction enzymes used for PCR-RFLP analysis.
460
3’SAG2 (VIII) GRA6 (X) 5’GRA7 (VIIа)
5’-ТCCTGTCAAGTTGTCTGCGG 5’-ATCCCCCTGTGCATCCAATA 5’-GAAATGTTTCAGGTTGCTGC 5’-GCAAGAGCGAACTTGAACAC 5’-ATTCTCATGCCTCCGCTTC 5’-AACGTTTCACGAAGGCACAC 5’-TTТCCGAGCAGGTGACCT 5’-TCGCCGAAGAGTTGACATAG 5’-ACCCTATATTGGGGCTTGCT 5’-TCGGTCTGAGACTGTCAACG 5’-TTCCGACGCTGAAGTGACTG 5’-ACACTGTCCTCGAGCTCCTA
461 Source 462
Dde I
[50]
Mbo I
[5,50]
Hha I Tru1 I
Eco R I Mbo II
[48]
[50]
Bse G I
Ac ce p
te
d
M
an
3’GRA7 (VIIа)
08(f) 010(r) А1 А2 B2 B1 GRA6(f) GRA6(r) GRA3 GRA4 GRA7 GRA2B
Restriction enzymes
ip t
5’SAG2 (VIII)
Primer sequence
cr
SAG1 (VIII)
Primer
us
Genetic marker (chr. location)
22
Page 22 of 26
462 463
Table 3 PCR program for each marker. 464
PCR program
SAG1 3 min 94°C 20 sec 94°C 40X 30 sec 56°C 30 sec 72°C 5 min 72°C
5’+3’ SAG2 5 min 94°C 20 sec 94°C 40X 30 sec 60°C 1 min 72°C 5 min 72°C
GRA6 5 min 95°C 45 sec 94°C 35X 1 min 56°C 1 min 72°C 10 min 72°C
5’+3’ GRA7 465 5 min 95°C 20 sec 94°C 466 40X 20 sec 60°C 30 sec 72°C 467 5 min 72°C
Ac ce p
te
d
M
an
us
cr
468 469
ip t
Marker
23
Page 23 of 26
Table 4 Isolation of T. gondii from animals. Method
Pigeons
Sheep
No. positive / No. analyzed
MAT
7/72
15/15
Real-Time PCR
5/7
2/12
Bioassay
4/4
1/1
ip t
469 470 471
cr
472 473
Ac ce p
te
d
M
an
us
474
24
Page 24 of 26
ip t
Amniotic fluid Blood Blood/ BALa
5’SAG2
3’SAG2
us
Clinical Entity SAG1
GRA6
5’GRA7
3’GRA7
I
I or II
I or III
I
I
Genotype
Reference
I
I or II
I or III
Reference
II or III
I or II
II
II
I or II
II
II or III
II
Reference
II or III
III
I or III
III
III
I or III
II or III
III
M an
Blood
Marker
Human (B)
Infection in pregnancy
II or III
I or II
II
II
I or II
II
II or III
II
Human (B)
CT (fetus)
II or III
I or II
II
II
I or II
II
II or III
II
Human (B)
CT (neonate) Reactivation after HSCT
NA
NA
NA
I
NA
NA
NA
Ib
II or III
I or II
II
II
I or II
I or III
II or III
Atypical
Human (D) Sheep (B)
Heart
Pigeon (B)
Heart
Pigeon (B)
Heart
Pigeon (B)
II or III
I or II
II
II
I or II
II
II or III
II
II or III
I or II
II
II
NA
II
II or III
II
II or III
III
I or III
III
III
I or III
II or III
III
II or III
I or II
II
II
I or II
II
II or III
II
Ac
Heart
ed
RH Type I Me49 Type II NED Type III
Origin (typing method)
ce pt
Sample type
cr
Table 5 PCR-RFLP genotyping of human and animal Toxoplasma gondii isolates from Serbia.
NA – not amplified BAL – bronchoalveolar lavage fluid a samples from the same patient b based on a single allele identified at the GRA6 locus B – genotyping performed after bioassay D – genotyping performed directly from the material
Page 25 of 26
Figure 1.
Ac ce p
te
d
M
an
us
cr
ip t
100 km
Page 26 of 26
S0147-9571(14)00018-6 http://dx.doi.org/doi:10.1016/j.cimid.2014.03.001 CIMID 961
To appear in: Received date: Revised date: Accepted date:
5-11-2013 27-2-2014 3-3-2014
ˇ Please cite this article as: Markovi´c M, Ivovi´c V, Stajner T, Djoki´c V, Klun I, Bobi´c B, Nikoli´c A, Djurkovi´c-Djakovi´c O, Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia, Comparative Immunology, Microbiology and Infectious Diseases (2014), http://dx.doi.org/10.1016/j.cimid.2014.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ORIGINAL ARTICLE
2 3 4
6
ip t
5
Evidence for genetic diversity of Toxoplasma gondii in selected
8
intermediate hosts in Serbia
9
us
10
cr
7
11
Marija Marković, Vladimir Ivović, Tijana Štajner, Vitomir Djokić, Ivana Klun,
13
Branko Bobić, Aleksandra Nikolić, Olgica Djurković-Djaković*
an
12
14
National Reference Laboratory for Toxoplasmosis, Institute for Medical Research,
16
University of Belgrade, Dr. Subotića 4, P.O. Box 102, 11129 Belgrade, Serbia
M
15
20 21 22 23 24 25 26 27
te
19
Running title: Toxoplasma genotypes in Serbia
Ac ce p
18
d
17
28 29 30 31 32
* Corresponding author. Tel.: +381 11 2685 788; fax: +381 11 2643 691. E-mail address: [email protected] (O. Djurković-Djaković).
33 1
Page 1 of 26
34
ABSTRACT
36
To contribute to the insight into the worldwide population structure of Toxoplasma gondii, we
37
genetically characterized a total of eight strains isolated from intermediate hosts including
38
humans, sheep and pigeons in Serbia. Although parasite DNA was detected in 28.2% (60/213)
39
of the human samples from 162 patients serologically suspected of active toxoplasmosis, as
40
well as in 5/7 seropositive pigeons and in 2/12 seropositive sheep examined, multilocus PCR-
41
RFLP genotyping, using SAG1, 5’SAG2, 3’SAG2, GRA6, 5’GRA7 and 3’GRA7 as markers,
42
was successful in only four human isolates (of which one was isolated from both the
43
bronchoalveolar lavage fluid and blood samples of a single patient), one sheep and three
44
pigeons. Of the eight isolates, five were type II (62.5%), one was type III, one was atypical,
45
and one had a type I allele at GRA6 as the single locus genotyped. Although type II, as
46
elsewhere in Europe, predominated, these results may suggest a higher genetic diversity of T.
47
gondii in Serbia, reflecting local environmental contamination and also the geographical
48
position of the country in South-East Europe. deletion
cr
us
an
M
d
te
Ac ce p
49 50
ip t
35
51
Key words: Toxoplasma gondii, intermediate hosts, humans, sheep, pigeons, genotyping,
52
PCR-RFLP, Serbia, South-East Europe
53 54 55 56 57
2
Page 2 of 26
57
Introduction Toxoplasma gondii, a ubiquitous protozoan, infects a broad range of mammals and birds
59
which all act as intermediate hosts, whereas felids are the only definitive hosts. In
60
intermediate hosts, following a brief acute stage characterized by the presence of circulating
61
tachyzoites, parasites convert to tissue cysts mainly localized in the muscular and neural
62
tissues. In felids, sexual reproduction of the parasite results in the production of oocysts shed
63
with feces, thereby contaminating the environment. Infection with either parasite form is
64
mainly peroral, and consumption of meat containing tissue cysts and ingestion of oocysts
65
from water and soil (through gardening and farming) have been shown to be major routes of
66
human infection [1,2]. Toxoplasmosis in humans is generally benign, but may have serious
67
consequences in the developing fetus in case of maternal infection in pregnancy and in
68
immunosuppressed individuals. In animals, T. gondii infection can cause various health
69
problems, ranging from mild to serious infectious disease symptoms to reproductive issues,
70
and even disseminated infection and death deletion [3]. The risk to human and animal health,
71
in line with the One Health concept, warrants evaluation of T. gondii distribution in the
72
environment. As the detection of T. gondii oocysts in water and soil is cumbersome and not
73
yet feasible on a large scale, assessing the presence of the parasite in its herbivore
74
intermediate hosts is a good indicator of environmental contamination.
Ac ce p
te
d
M
an
us
cr
ip t
58
75
The population structure of T. gondii is unexpectedly oligoclonal for a parasite whose life
76
cycle involves sexual reproduction. Initially, it was characterized by three main clonal
77
lineages designated as type I, II and III that were found, with the predominance of type II, in
78
Europe and North America [4-6]. However, as data for South America and Africa became
79
available, a higher frequency of non-clonal strains (atypical and recombinant) was revealed,
80
indicating that the population structure is more diverse than previously thought [7-10].
81
Moreover, specific African non-clonal genotypes, termed Africa 1, 2 and 3, have been 3
Page 3 of 26
82
identified [11, 12]. But the picture is currently being complicated in the Western World as
83
well, since a fourth clonal lineage has been recently described in North America [13]. Strain genotype has been associated with clinical severity of human toxoplasmosis [4].
85
Type II strains have been shown to be most prevalent in congenital infection and AIDS
86
patients in North America and Europe [5,6,14]. Atypical strains have been associated with
87
severe toxoplasmosis in immunocompetent patients [15,16], particularly in South America
88
where they correlate with ocular toxoplasmosis [7], and in the setting of immunosuppression
89
[17]. In animal hosts, emerging information indicates severe histopathological lesions in
90
sheep abortions caused by T. gondii of an atypical genotype [18], but also the association of
91
type II strains with fatal toxoplasmosis in a cat [19], arctic foxes [20] and wild hares [21].
an
us
cr
ip t
84
Information on the population structure of T. gondii is important in view of the possible
93
health implications. However, data are quite scarce for South-East Europe, Serbia included,
94
where the genotype of a single human isolate has been reported [22] and virtually none in
95
animals. The aim of this study was thus to identify T. gondii genotypes circulating in Serbia
96
by detecting the parasite in three of its intermediate hosts.
97
1. Materials and Methods
98
2.1. Study area
Ac ce p
te
d
M
92
99
Located in South-East Europe, Serbia (without Kosovo) extends over a territory of 77,512
100
km2 and has a population of 7.5 million. Belgrade with its surroundings (Belgrade District) is
101
a highly urban region, with a population of approximately 1.6 million (Fig. 1A).
102
2.2. Collection of samples
103
Genotyping was attempted from both human and animal biological materials.
4
Page 4 of 26
Human materials involved a total of 213 body fluid samples (Table 1) from 162 patients
105
both clinically and serologically suspected of active toxoplasmosis, defined as infection stages
106
characterized by the presence of tachyzoites in body fluids. These included adults suspected
107
of acute toxoplasmosis, women suspected of infection during pregnancy and neonates
108
suspected of congenital toxoplasmosis (CT), patients with ocular and cerebral toxoplasmosis
109
and patients who underwent hematopoietic stem cell transplantation (HSCT), who were
110
referred to the National Reference Laboratory for Toxoplasmosis (NRLToxo) between 2008
111
and 2012. Since NRLToxo is the single laboratory in Serbia to perform expert diagnosis,
112
patients originated from all over the country.
us
cr
ip t
104
Animals tested were sheep (Ovis aries) and pigeons (Columba livia) and materials
114
included blood samples and hearts. Sheep samples from a total of 15 ewes (mean age 14.7
115
months, range 13-24 months), were collected from two abattoirs in the vicinity of Belgrade
116
(in the administrative municipalities of Pećinci and Stara Pazova), which serve the local farms
117
(Fig. 1B). All sheep were destined for human consumption. Pigeons were collected within the
118
scope of an ongoing large ecological investigation of feral pigeons as indicators of
119
environmental contamination led by the Natural History Museum in Belgrade. For this study,
120
hearts and blood samples from 72 pigeons captured at 15 collection sites, mainly in downtown
121
Belgrade (Fig. 1B) were made available to us.
122
2.3. Serology
Ac ce p
te
d
M
an
113
123
Blood samples were initially examined for T. gondii specific antibodies. Human samples
124
were tested for specific IgG and IgM antibodies and avidity of specific IgG antibodies using
125
commercial tests on a VIDAS analyzer (bioMérieux, Marcy l’Etoile, France). Positivity
126
thresholds were 8 IU/mL for IgG and 0.65 for IgM, while avidity was expressed in index
127
values (low: I < 0.2; intermediate: 0.2 ≤ I < 0.3; high: I ≥ 0.3). In some cases, specific IgM 5
Page 5 of 26
antibodies were additionally tested by the immunosorbent agglutination assay (ISAgA,
129
bioMérieux, index scale 0-12, positivity threshold I = 9). Commercial assays were performed
130
according to the manufacturer’s instructions. Specific IgG antibodies were also tested by an
131
in-house high sensitivity agglutination test (cut-off value = 1:20) [23]. A slight adaptation of
132
this test (the modified agglutination test, MAT) was used for animal samples [24]. Sheep and
133
pigeon sera were serially two-fold diluted starting at 1:10 and sera reactive at ≥ 1:10 were
134
considered positive [25]. In bioassay experiments, the cut-off was set at 1:20.
135
2.4. Study design
us
cr
ip t
128
In human biological materials obtained from patients serologically suspected of active
137
toxoplasmosis, genotyping was attempted both directly and, in an attempt to isolate and
138
propagate the parasite, following a bioassay step. In animal samples, DNA was extracted from
139
the hearts of seropositive sheep and pigeons following trypsin digestion, and PCR-positive
140
hearts were further bioassayed in mice.
M
d
The study followed the tenets of the Declaration of Helsinki and was approved by a local
te
141
an
136
(Institute for Medical Research) Ethics Committee.
143
2.5. Trypsin digestion of animal tissue
Ac ce p
142
144
Whole animal hearts (sheep ! 200g, pigeon 5-6 g) were mixed in an electric blender and
145
incubated at 37 °C for 1.5 h with trypsin (final concentration 0.25%) and antibiotics [peni-
146
strepto (PAA Laboratories GmbH, Pasching, Austria) and amoxicillin (Hemofarm, Vršac,
147
Serbia)]. The suspension was then filtered and washed with saline three times. The obtained
148
pellet was resuspended in saline, to 2 mL for sheep and to 1 mL for pigeon material. Part (200
149
! L) of the homogenated tissue was analyzed by PCR and the remaining tissue of the positive
150
samples was bioassayed.
151
2.6. Bioassay 6
Page 6 of 26
Bioassay was performed by intraperitoneal inoculation of suspected material (! 500 ! L),
153
with addition of gentamycin (Galenika, Zemun, Serbia), into two Swiss-Webster female mice
154
per sample, as previously described [26]. After six weeks the mice were euthanized, blood
155
was tested by MAT and brains were homogenized with 1 mL of saline each for microscopic
156
examination for T. gondii cysts. Cysts were enumerated at a sensitivity threshold of 10 cysts
157
per brain. A bioassay was considered positive in case of positive serology, detection of brain
158
cysts, or both, and DNA was extracted from the brains of all positive mice.
159
2.7. DNA extraction
us
cr
ip t
152
DNA was extracted from 200 ! l of biological materials using the QIAmp DNA mini kit
161
(Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Extracted
162
DNA was resuspended in 150 ! l of nuclease-free water and stored at -20°C.
163
2.8. Detection of T. gondii DNA by Real-Time PCR
d
M
an
160
T. gondii DNA was detected by Real-Time PCR (RT-PCR) targeted at the 529 bp
165
repetitive element (gene bank accession number AF146527), as previously described [27].
166
Briefly, the PCR reaction was performed in a final volume of 20 μL mixture containing 10 μl
167
Maxima™ Probe/ROX qPCR Master Mix (2X) [Fermentas (Thermo Fisher Scientific),
168
Waltham, MA, USA], 0.25 mM of each primer, 0.10 mM of TaqMan probe (Invitrogen, Life
169
Technologies, Carlsbad, CA, USA), 0.015 U/μL of UNG, 25 mM MgCl2 and nuclease-free
170
water, plus 3 μL of extracted DNA. Amplification was performed over 40 cycles: 2 min at 50
171
ºC for UNG pre-treatment, 10 min at 94 ºC initial denaturation, followed by 40 cycles of 15 s
172
at 95 ºC for denaturation and 60 s at 60 ºC for annealing/extension.
173
2.9. Genotype analysis of T. gondii by multilocus PCR-RFLP
Ac ce p
te
164
7
Page 7 of 26
Genotyping was performed by the PCR-restriction fragment length polymorphism (PCR-
175
RFLP) method using six markers including SAG1, 5’SAG2, 3’SAG2, GRA6, 5’GRA7 and
176
3’GRA7, across four genetic loci (Table 2). For each marker the PCR reaction mixture
177
consisted of 12.5 μL PCR Master Mix (2X) (Fermentas), 1 μL 10 μМ forward and reverse
178
primer, 7.5 μL nuclease free water and 3 μL DNA extracted from the sample in a 25 μL
179
reaction volume. For each marker the PCR program was different (Table 3). Positive controls
180
consisted of T. gondii type I (RH), type II (Me49) and type III (NED) strains, while nuclease
181
free water was used as a negative control. To confirm DNA amplification, PCR products were
182
separated by electrophoresis in 3% agarose gel, and digested with appropriate restriction
183
enzymes for different markers. The PCR mixture for digestion consisted of 12 μL of nuclease-
184
free water, 2.5 μL of buffer, 0.5 μL of restriction enzyme (concentration 5 U/µL for MbO II,
185
10 U/µL for all others), and 10 μL of PCR product. RFLPs were visualized by electrophoresis
186
in 3% agarose gel stained with ethidium bromide. Estimation of fragment size was based on
187
comparison to a 50 bp DNA ladder (Fermentas).
188
2. Results
Ac ce p
te
d
M
an
us
cr
ip t
174
189
A total of 213 biological samples from 162 patients serologically suspected of active
190
toxoplasmosis was tested by RT-PCR. T. gondii DNA was detected in 60 (28.2%) samples
191
from 52 individuals (Table 1). Of the 132 bioassay experiments, 30 were positive for cysts
192
and/or serologically.
193
T. gondii-specific antibodies were found in all tested sheep. DNA was extracted from the
194
hearts of 12 animals, of which two were RT-PCR positive. Due to technical reasons, only one
195
was bioassayed, leading to the isolation of a T. gondii strain (Table 4).
196
Seven of the 72 pigeons (9.7%, 95% CI 2.9-16.5) were seropositive for T. gondii and DNA
197
was extracted from the hearts of all seropositive animals. T. gondii DNA was detected in five 8
Page 8 of 26
198
animals, of which four were bioassayed and all four bioassays were positive (three based on
199
the detection of cysts of which one at an extremely high cyst burden of ~2000 cysts per brain;
200
one only by MAT) (Table 4). Based on previous experience in our laboratory that genotyping is feasible only in samples
202
which are RT-PCR positive at a cycle threshold (Ct) value of below 30, it was attempted in a
203
total of 19 human samples. However, as many of those were actually PCR-positive at a Ct
204
close to 30, and the harvest of brain cysts was poor in many bioassay experiments, genotypes
205
of four human isolates were determined. Of these, two belonged to type II, one was non-
206
clonal and one, typed only on GRA6, had a type I allele (Table 5). The non-clonal strain,
207
isolated from the blood and BAL fluid of a single patient, was type II according to SAG1,
208
5’SAG2, 3’SAG2 and GRA6, but had alleles of both type I and II on 5’GRA7 and 3’GRA7,
209
indicating that it could be atypical, which was later confirmed by microsatellite analysis [17].
210
This strain was also the single one directly typed from both the blood and BAL samples.
d
M
an
us
cr
ip t
201
Genotyping of animal isolates was attempted from strains isolated by bioassay from four
212
pigeons and one sheep and was successful in three pigeons and one sheep; of the three T.
213
gondii isolates from pigeons two were type II and one was type III, while the strain isolated
214
from sheep belonged to the type II lineage (Table 5).
215
3. Discussion
Ac ce p
te
211
216
Genotyping of T. gondii isolates in Serbia revealed evidence of the presence of several
217
genetic types. Of the eight T. gondii strains isolated from three intermediate host species
218
(humans, sheep and pigeons), type II was predominant (62.5%, 5/8), but one (12.5%)
219
belonged to type III, one was atypical, and one, only genotyped at the GRA6 locus, had a type
220
I allele. The latter finding, however, should be taken with caution; using only GRA6 which
221
may distinguish among clonal lineages was at one time considered to be sufficient for 9
Page 9 of 26
222
European strains where only these are expected [28], typing of a single allele does not
223
necessarily imply that all alleles are of the same type [29]. Isolation of these different genotypes among eight isolates in a rather small territory may
225
suggest a greater genetic diversity than would be expected from other studies in Europe. In
226
humans, studies in France showed a large predominance of type II isolates (>80%), with the
227
occurrence of some type I and type III isolates [6,14]. Few studies of human isolates have
228
been performed elsewhere in Europe. Nowakowska et al. [30] reported that among cases of
229
CT in Poland, all nine genotyped isolates were type II. Two human strains isolated from CT
230
cases in Serbia and recently in the neighboring Romania, both belonged to type II [22,31].
us
cr
ip t
224
Type II is also vastly predominant in animals in Europe, such as in chicken in Austria [32],
232
in cats in Germany [33], and even in wildlife such as carnivorous mammals [34], rodents and
233
foxes [35], arctic foxes [20] and hares [21]. In this study, we analyzed sheep and pigeons as
234
species that may be considered markers of environmental contamination. Sheep are
235
herbivores and as such get infected mainly by ingesting T. gondii oocysts by grazing on
236
pastures or through contaminated water or feed. All 15 sheep analyzed in this study were
237
previously exposed to T. gondii as shown by 100% seropositivity, and the single genotyped
238
isolate was characterized as type II. This is in agreement with previous work on sheep. In a
239
study in England where isolation of T. gondii was performed from ovine abortion tissue, all
240
13 isolated strains belonged to type II [36]. In France, analysis of eight and 46 strains isolated
241
from ovine meat destined for human consumption, showed that all were type II except a
242
single one which belonged to type III [37,38].
Ac ce p
te
d
M
an
231
243
Pigeons are free range animals that also get infected feeding from the ground. Pigeons live
244
in close association with humans and their daily migrations are relatively limited. It has been
245
shown that, in search for food, they rarely cross distances over 0.5-0.6 km [39], reflecting
246
environmental contamination of the particular area where they are captured. And as prey to 10
Page 10 of 26
cats, pigeons may also contribute to environmental dissemination of T. gondii. Few studies
248
have analyzed T. gondii genotypes of isolates from birds in general; of the 12 genotyped
249
pigeon isolates in Portugal, Waap et al. [40] showed that nine were type II, but two type III
250
and one type I were isolated as well. We here report that among the three genotyped pigeon
251
isolates in Serbia, two were type II while one was type III. On the other hand, predominance
252
of type III over type II isolates was shown in several bird species in Egypt and Iran [41,42].
cr
ip t
247
The described vast predominance of type II among both human and animal isolates
254
elsewhere in Europe does not seem to apply to the Mediterranean, where type III in particular
255
was much more frequent, detected in as many as 80% of the human and rodent isolates in the
256
islands of Cyprus and Crete [43]; and in 33%, 26% and 16%, respectively, of the strains
257
isolated from chickens, pigs and pigeons in Portugal [44,45,40]. In Spain, out of 25 human
258
isolates, type I, II and III have been detected at a ratio of 40, 40, and 20%, respectively [46].
259
Since these studies (with the exception of Waap et al. [40] who used five microsatellite
260
markers) were based on the detection of a single marker, i.e. GRA6 or SAG2, which although
261
capable of differentiating among the three clonal types, can be insufficient to detect and
262
resolve atypical alleles from type III and to a lesser extent, from type I [28,47,48], it is
263
possible that there may have been atypical strains among those reported as type III; if so, the
264
Mediterranean area may be characterized by a larger genetic diversity compared to
265
continental Europe.
Ac ce p
te
d
M
an
us
253
266
Since Serbia borders with the Mediterranean area, it may not be surprising that, as
267
presented here, intermediate hosts harbored genetically diverse strains. Use of additional
268
genetic markers to the six ones we used may possibly identify other alleles, characterizing
269
non-clonal strains, thereby even increasing the observed diversity. The presented T. gondii
270
genetic diversity may be all the more important since it has been observed in a relatively
271
small geographical area. The findings plausibly reflect the geographical position of Serbia in 11
Page 11 of 26
South-East Europe, in the centre of the Balkan Peninsula, an intersection between Europe and
273
Asia, and in relative proximity to Africa. Similarly, the recent finding of the Africa 1
274
genotype in two human isolates from Turkey was associated with the geographical position of
275
Turkey between Asia, Europe and Africa [49]. A higher genetic diversity including the
276
presence of non-clonal T. gondii in Serbia may indicate phylogenetic ties among Asian,
277
African and European T. gondii populations, or, in evolutionary terms more recent
278
environmental contamination which may occur from migratory birds that often stopover in
279
South-East Europe during their seasonal movement from Africa to the North. A relatively
280
novel factor that may contribute to T. gondii genetic diversity involves globalization of food
281
including importation of meats from areas of a highly divergent population structure.
282
Irrespective of the origin, the public health and veterinary implications of the genetic diversity
283
of T. gondii warrant more research in this and other areas of the world to fully appreciate the
284
organism’s population structure and its ramifications.
285
287
288 289 290
Conflict of interests
The authors declare no conflict of interests.
Ac ce p
286
te
d
M
an
us
cr
ip t
272
Acknowledgements
The results of this paper have been presented in part at the 11th European Multicolloquium of Parasitology (EMOP XI), Cluj-Napoca, Romania, 25-29 July, 2012. deletion
291
The authors are grateful to Isabelle Villena (Reims, France) for kindly supplying the
292
antigen for HSDA, and the reference DNA for type III (NED strain). We also wish to
293
acknowledge Marko Raković, Natural History Museum of Belgrade, for kindly letting us the
294
pigeon samples.
12
Page 12 of 26
This study was supported by a grant (project No. III 41019) from the Ministry of
295
Education, Science and Technological Development of Serbia.
297
References
298
[1]
ip t
296
Bobić B, Jevremović I, Marinković J, Šibalić D, Djurković-Djaković O. Risk factors for Toxoplasma infection in a reproductive age female population in the area of Belgrade,
300
Yugoslavia. Eur J Epidemiol 1998;14:605-10. [2]
Cook AJC, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, et al. on behalf
us
301
cr
299
of the European Research Network on Congenital Toxoplasmosis. Sources of
303
toxoplasma infection in pregnant women: European multicentre casecontrol study. Brit
304
Med J 2000;321:142-7.
an
302
[3]
Dubey JP. Toxoplasmosis of animals and humans. CRC Press, Boca Raton, FL; 2010.
306
[4]
Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation
1997;35:1411-4.
310
[6]
Ajzenberg D, Cogné N, Paris L, Bessières MH, Thulliez P, Filisetti D, et al. Genotype
of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and
312
correlation with clinical findings. J Infect Dis 2002;186:684-9.
313 314
Ac ce p
Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol
309
311
Howe DK, Honore S, Derouin F, Sibley LD. Determination of genotypes of
te
[5]
d
of parasite genotype with human disease. J Infect Dis 1995;172:1561-6.
307 308
M
305
[7]
Khan A, Jordan C, Muccioli C,Vallochi AL, Rizzo LV, Belfort Jr R, et al. Genetic
315
divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil.
316
Emerg Infect Dis 2006;12:942-9.
13
Page 13 of 26
317
[8]
Al-Kappany YM, Rajendran C, Abu-Elwafa SA, Hilali M, Su C, Dubey JP. Genetic
318
diversity of Toxoplasma gondii isolates in Egyptian feral cats reveals new genotypes. J
319
Parasitol 2010;96:1112-4. [9]
Ferreira IM, Vidal JE, de Mattos Cde C, de Mattos LC, Qu D, Su C, Pereira-Chioccola
ip t
320
VL. Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human
322
patients in Sao Paulo State, Brazil identified distinct genotypes. Exp Parasitol
323
2011;129:190-5.
cr
321
[10] Demar M, Hommel D, Djossou F, Peneau C, Boukhari R, Louvel D, et al. Acute
325
toxoplasmosis in immunocompetent patients hospitalized in an intensive care unit in
326
French Guiana. Clin Microbiol Infect 2012;18:221-31.
an
us
324
[11] Ajzenberg D, Bañuls AL, Su C, Dumètre A, Demar M, Carme B, Dardé ML. Genetic
328
diversity, clonality and sexuality in Toxoplasma gondii. International J Parasitol
329
2004;34: 1185-96.
d
M
327
[12] Mercier A, Devillar S, Ngoubangoye B, Bonnabau H, Bañuls AL, Durand P, et al.
331
Additional haplogroups of Toxoplasma gondii out of Africa: population structure and
332
mouse-virulence of strains from Gabon. PLOS Negl Trop Dis 2010;4,876.
Ac ce p
te
330
333
[13] Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD. Genetic analyses of
334
atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Int J
335
Parasitol 2011;41:645-55.
336
[14] Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, et al. Genotype of 88
337
Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised
338
patients and correlation with clinical findings. J Infect Dis 2009;199:1155-67.
339
[15] Carme B, Bissuel F, Ajzenberg D, Bouyne R, Aznar C, Demar M, et al. Severe acquired
340
toxoplasmosis in immunocompetent adult patients in French Guiana. J Clin Microbiol
341
2002;40:4037-44. 14
Page 14 of 26
342
[16] Delhaes L, Ajzenberg D, Sicot B, Bourgeot P, Dardé ML, Dei-Cas E, Houfflin-Debarge
343
V. Severe congenital toxoplasmosis due to a Toxoplasma gondii strain with an atypical
344
genotype: case report and review. Prenat Diagn 2010;30:902-5. [17] Štajner T, Vasiljević Z, Vujić D., Marković M, Ristić G, Mićić D, et al. Atypical strain
346
of Toxoplasma gondii causing fatal reactivation after haematopoietic stem cell
347
transplantion in a patient with an underlying immunological deficiency. J Clin
348
Microbiol 2013; 51, 2686-90.
cr
ip t
345
[18] Edwards JF, Dubey JP. Toxoplasma gondii abortion storm in sheep on a Texas farm and
350
isolation of mouse virulent atypical genotype T. gondii from an aborted lamb from a
351
chronically infected ewe.Vet Parasitol. 2013;192,129-36.
an
us
349
[19] Spycher A, Geigy C, Howard J, Posthaus H, Gendron K, Gottstein B, et al. Isolation
353
and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an
354
immunocompetent 10-year-old cat. J Vet Diagn Invest. 2011;23,104-8.
d
M
352
[20] Prestrud KW, Asbakk K, Mørk T, Fuglei E, Tryland M, Su C. Direct high-resolution
356
genotyping of Toxoplasma gondii in arctic foxes (Vulpes lagopus) in the remote arctic
357
Svalbard archipelago reveals widespread clonal Type II lineage.Vet Parasitol.
Ac ce p
358
te
355
2008;158:121-8.
359
[21] Jokelainen P, Isomursu M, Näreaho A, Oksanen A. Natural Toxoplasma gondii
360
infections in European brown hares and mountain hares in Finland: proportional
361 362
mortality rate, antibody prevalence, and genetic characterization. J Wildl Dis. 2011;47:154-63.
363
[22] Djurković-Djaković O, Klun I, Khan A, Nikolić A, Knezević-Ušaj S, Bobić B, Sibley
364
LD. A human origin type II strain of Toxoplasma gondii causing severe encephalitis in
365
mice. Microb Infect 2006;8:2206-12.
15
Page 15 of 26
366
[23] Desmonts G, Remington JS. Direct agglutination test for diagnosis of Toxoplasma
367
infection: method for increasing sensitivity and specificity. J Clin Microbiol
368
1980;11:562-8.
370
[24] Dubey JP, Desmonts G. Serological responses of equids fed Toxoplasma gondii ocysts.
ip t
369
Eq Vet J 1987;19:337-9.
[25] Dubey JP, Graham DH, Blackston CR, Lehmann T, Gennari SM, Ragozo AM, et al.
372
Biological and genetic characterization of Toxoplasma gondii isolates from chickens
373
(Gallus domesticus) from São Paulo, Brazil: unexpected findings. Int J Parasitol
374
2002;32:99-105.
us
cr
371
[26] Djurković-Djaković O, Nikolić A, Bobić B, Klun I, Aleksić A. Stage conversion of
376
Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine
377
dithiocarbamate. Microb Infect 2005;7:49-54.
M
an
375
[27] Vujanić M, Ivović V, Kataranovski M, Nikolić A, Bobić B, Klun I, et al.
379
Toxoplasmosis in naturally infected rodents in Belgrade, Serbia. Vector Borne Zoon Dis
380
2011;11:1209-11.
382 383 384
te
[28] Fazaeli A, Carter PE, Darde ML, Pennington TH. Molecular typing of Toxoplasma
Ac ce p
381
d
378
gondii strains by GRA6 gene sequence analysis. Int J Parasitol 2000;30:637-42.
[29] Ajzenberg D. Type I strains in human toxoplasmosis: myth or reality? Future Microbiol. 2010;5:841-3.
385
[30] Nowakowska D, Colon I, Remington JS, Grigg M, Golab E, Wilczynski J, Sibley LD.
386
Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological
387
testing of samples from infants in Poland diagnosed with congenital toxoplasmosis. J
388
Clin Microbiol 2006;44:1382-9.
16
Page 16 of 26
389
[31] Costache CA, Colosi HA, Blaga L, Györke A, Pastiu AI, Colosi IA, Ajzenberg D. First
390
isolation and genetic characterization of a Toxoplasma gondii strain from a symptomatic
391
human case of congenital toxoplasmosis in Romania. Parasite 2013;20:11. [32] Dubey JP, Edelhofer R, Marcet P, Vianna MC, Kwok OC, Lehmann T. Genetic and
393
biologic characteristics of Toxoplasma gondii infections in free-range chickens from
394
Austria. Vet Parasitol 2005;133:299-306.
cr
ip t
392
[33] Herrmann DC, Pantchev N, Globokar Vrhovec M, Barutzki D, Wilking H, Fröhlich A,
396
et al. Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in
397
Germany. Int J Parasitol 2010;40:285-92.
us
395
[34] Burrells A, Bartley PM, Zimmer IA, Roy S, Kitchener AC, Meredith A, et al.
399
Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian
400
carnivores
401
10.1017/S0031182013001169.
the
UK.
Parasitology
2013
[Epub
ahead
of
print].
doi:
d
in
M
an
398
[35] Herrmann DC, Maksimova P, Maksimova A, Sutora A, Schwarza S, Jaschkeb W, et al.
403
Toxoplasma gondii in foxes and rodents from the German Federal States of
404
Brandenburg and Saxony-Anhalt: Seroprevalence and genotypes. Vet Parasitol
406 407
Ac ce p
405
te
402
2012;185:78-85.
[36] Owen MR, Trees AJ. Genotyping of Toxoplasma gondii associated with abortion in sheep. J Parasitol 1999;85:382-4.
408
[37] Dumetre A, Ajzenberg D, Rozette L, Mercier A, Dardé ML. Toxoplasma gondii
409
infection in sheep from Haute-Vienne, France: seroprevalence and isolate genotyping
410
by microsatellite analysis. Vet Parasitol 2006;142:376-9.
411
[38] Halos L, Thebault A, Aubert D, Thomas M, Perret C, Geers R, et al. An innovative
412
survey underlining the significant level of contamination by Toxoplasma gondii of
413
ovine meat consumed in France. Int J Parasitol 2010;40:193-200. 17
Page 17 of 26
414 415
[39] Rose E, Nagel P, Haag-Wackernagel D. Spatio-temporal use of the urban habitat by feral pigeons (Columba livia). Behav Ecol Sociobiol 2006;60:242-54. [40] Waap H, Vilares A, Rebelo E, Gomes S, Angelo H. Epidemiological and genetic
417
characterization of Toxoplasma gondii in urban pigeons from the area of Lisbon
418
(Portugal). Vet Parasitol 2008;157:306-9.
ip t
416
[41] Dubey JP, Graham DH, Dahl E, Hilali M, El-Ghaysh A, Sreekumar C, et al. Isolation
420
and molecular characterization of Toxoplasma gondii from chickens and ducks from
421
Egypt. Vet Parasitol 2003;114:89-95.
us
cr
419
[42] Khademvatan S, Saki J, Yousefi E, Abdizadeh R. Detection and genotyping of
423
Toxoplasma gondii strains isolated from birds in the southwest of Iran. Brit Poultry Sci
424
2013;54:76-80.
M
an
422
[43] Messaritakis I, Detsika M, Koliou M, Sifakis S, Antoniou M. Prevalent genotypes of
426
Toxoplasma gondii in pregnant women and patients from Crete and Cyprus. Amer J
427
Trop Med Hyg 2008;79:205-9.
te
d
425
[44] Dubey JP, Vianna MC, Sousa S, Canada N, Meireles S, Correia da Costa JM, et al.
429
Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal. J
430
Ac ce p
428
Parasitol 2006;92:184-6.
431
[45] de Sousa S, Ajzenberg D, Canada N, Freire L, da Costa JM, Darde ML, et al. Biologic
432
and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.
433
Vet Parasitol 2006;135:133-7.
434
[46] Fuentes I, Rubio JM, Ramirez C, Alvar J. Genotypic characterization of Toxoplasma
435
gondii strains associated with human toxoplasmosis in Spain: direct analysis from
436
clinical samples. J Clin Microbiol 2001;39:1566-70.
437 438
[47] Ajzenberg D, Dumètre A, Dardé ML. Multiplex PCR for typing strains of Toxoplasma gondii. J Clin Microbiol 2005;43:1940-3. 18
Page 18 of 26
439
[48] Su C, Zhang X, Dubey JP. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP
440
markers: a high resolution and simple method for identification of parasites. Int J
441
Parasitol 2006;36:841-8. [49] Döşkaya M, Caner A, Ajzenberg D, Değirmenci A, Dardé ML, Can H, et al. Isolation of
443
Toxoplasma gondii strains similar to Africa 1 genotype in Turkey. Parasitol Int
444
2013;62,471-4.
cr
ip t
442
[50] Richomme C, Aubert D, Gilot-Fromont E, Ajzenberg D, Mercier A, Ducrot C, et al.
446
Genetic characterization of Toxoplasma gondii from wild boar (Sus scrofa) in France.
447
Vet Parasitol. 2009;164:296-300.
us
445
Ac ce p
te
d
M
an
448
19
Page 19 of 26
448
LEGEND FOR FIGURE
449 450
FIG. 1. A: Map of Europe with Serbia shaded grey. B: Map of Serbia (Belgrade District in
452
black),
sheep collection site,
ip t
451
pigeon collection site; source: www.wikimedia.org.
Ac ce p
te
d
M
an
us
cr
453
20
Page 20 of 26
453
Peripheral blood Fetal blood Amniotic fluid Aqueous humor Cerebrospinal fluid BAL ∑
No. examined 138 14 38 12 10 1 213
% positive 24.6 21.4 28.9 50.0 50.0 100.0 28.2
te
d
M
an
BAL – bronchoalveolar lavage fluid
Ac ce p
456 457 458
RT-PCR No. positive 34 3 11 6 5 1 60
cr
Sample type
ip t
Table 1 Toxoplasma gondii DNA detection in human biological materials.
us
454 455
21
Page 21 of 26
458
Table 2
459
Genetic markers, primer sequences and restriction enzymes used for PCR-RFLP analysis.
460
3’SAG2 (VIII) GRA6 (X) 5’GRA7 (VIIа)
5’-ТCCTGTCAAGTTGTCTGCGG 5’-ATCCCCCTGTGCATCCAATA 5’-GAAATGTTTCAGGTTGCTGC 5’-GCAAGAGCGAACTTGAACAC 5’-ATTCTCATGCCTCCGCTTC 5’-AACGTTTCACGAAGGCACAC 5’-TTТCCGAGCAGGTGACCT 5’-TCGCCGAAGAGTTGACATAG 5’-ACCCTATATTGGGGCTTGCT 5’-TCGGTCTGAGACTGTCAACG 5’-TTCCGACGCTGAAGTGACTG 5’-ACACTGTCCTCGAGCTCCTA
461 Source 462
Dde I
[50]
Mbo I
[5,50]
Hha I Tru1 I
Eco R I Mbo II
[48]
[50]
Bse G I
Ac ce p
te
d
M
an
3’GRA7 (VIIа)
08(f) 010(r) А1 А2 B2 B1 GRA6(f) GRA6(r) GRA3 GRA4 GRA7 GRA2B
Restriction enzymes
ip t
5’SAG2 (VIII)
Primer sequence
cr
SAG1 (VIII)
Primer
us
Genetic marker (chr. location)
22
Page 22 of 26
462 463
Table 3 PCR program for each marker. 464
PCR program
SAG1 3 min 94°C 20 sec 94°C 40X 30 sec 56°C 30 sec 72°C 5 min 72°C
5’+3’ SAG2 5 min 94°C 20 sec 94°C 40X 30 sec 60°C 1 min 72°C 5 min 72°C
GRA6 5 min 95°C 45 sec 94°C 35X 1 min 56°C 1 min 72°C 10 min 72°C
5’+3’ GRA7 465 5 min 95°C 20 sec 94°C 466 40X 20 sec 60°C 30 sec 72°C 467 5 min 72°C
Ac ce p
te
d
M
an
us
cr
468 469
ip t
Marker
23
Page 23 of 26
Table 4 Isolation of T. gondii from animals. Method
Pigeons
Sheep
No. positive / No. analyzed
MAT
7/72
15/15
Real-Time PCR
5/7
2/12
Bioassay
4/4
1/1
ip t
469 470 471
cr
472 473
Ac ce p
te
d
M
an
us
474
24
Page 24 of 26
ip t
Amniotic fluid Blood Blood/ BALa
5’SAG2
3’SAG2
us
Clinical Entity SAG1
GRA6
5’GRA7
3’GRA7
I
I or II
I or III
I
I
Genotype
Reference
I
I or II
I or III
Reference
II or III
I or II
II
II
I or II
II
II or III
II
Reference
II or III
III
I or III
III
III
I or III
II or III
III
M an
Blood
Marker
Human (B)
Infection in pregnancy
II or III
I or II
II
II
I or II
II
II or III
II
Human (B)
CT (fetus)
II or III
I or II
II
II
I or II
II
II or III
II
Human (B)
CT (neonate) Reactivation after HSCT
NA
NA
NA
I
NA
NA
NA
Ib
II or III
I or II
II
II
I or II
I or III
II or III
Atypical
Human (D) Sheep (B)
Heart
Pigeon (B)
Heart
Pigeon (B)
Heart
Pigeon (B)
II or III
I or II
II
II
I or II
II
II or III
II
II or III
I or II
II
II
NA
II
II or III
II
II or III
III
I or III
III
III
I or III
II or III
III
II or III
I or II
II
II
I or II
II
II or III
II
Ac
Heart
ed
RH Type I Me49 Type II NED Type III
Origin (typing method)
ce pt
Sample type
cr
Table 5 PCR-RFLP genotyping of human and animal Toxoplasma gondii isolates from Serbia.
NA – not amplified BAL – bronchoalveolar lavage fluid a samples from the same patient b based on a single allele identified at the GRA6 locus B – genotyping performed after bioassay D – genotyping performed directly from the material
Page 25 of 26
Figure 1.
Ac ce p
te
d
M
an
us
cr
ip t
100 km
Page 26 of 26