The occurrence of Toxoplasma gondii infection in people and animals from rural environment of Lublin region - estimate of potential role of water as a source of infection.
ABSTRACT A total of 254 humans and 489 domestic animals living on farms in the Lublin province (eastern Poland) were examined for the presence of antibodies against Toxoplasma gondii using, respectively, the ELFA and direct agglutination tests. In parallel, 182 samples of potable water, mostly from shallow household wells, were taken on farms and examined for the presence of T. gondii by microscopy and PCR. The frequency of seropositive reactions in farm inhabitants (66.9 percent) was significantly greater (p < 0.01) compared to the reference group of 39 healthy urban dwellers (41.0 percent). A highly significant positive correlation was found between the age of examined farm inhabitants and the rate of positive reactions with Toxoplasma antigen (p < 0.0001). Among domestic animals, the greatest frequency of seropositive reactions to T. gondii occurred in cats (75.0 percent) and dogs (53.6 percent), less frequent in cattle (33.8 percent) and hens (33.5 percent) and the least frequent in pigs (17.9 percent) and ducks (21.2 percent). The presence of T. gondii was found in potable water samples taken from water intakes on farms: in 12.6 percent of samples by microscopy, and in 22.5 percent of samples by PCR. Among 19 water samples taken from bathing places on the territory of the Lublin province, 2 samples positive for T. gondii (10.5 percent) were found by microscopic examination and confirmed by PCR . The presence of live parasites in water samples was demonstrated by the isolation of Toxoplasma gondii strains in mice. By use of RFLP -PCR it was found that the majority of isolated Toxoplasma strains (78.0 percent) belonged to clonal type I which is most virulent for humans and animals. Although no statistically significant relationship between the presence of T. gondii in water and occurrence of seropositive reactions in farm inhabitants and/or domestic animals could be found, the above-mentioned data suggest a potential role of potable water in the spread of toxoplasmosis in the rural environment.
- SourceAvailable from: Marie Laure Dardé[show abstract] [hide abstract]
ABSTRACT: The majority of Toxoplasma gondii strains from a variety of human and animal sources have been grouped into three highly clonal but closely related lineages. The low occurrence of nucleotide differences among the three predominant lineages and their unusual dimorphic allelic composition suggest that they have arisen from a recent common ancestry. Less than 1% of the previously studied strains contain unique genotypes and high divergence of DNA sequence, and therefore are considered 'exotic' or 'atypical' strains. The seemingly low genetic diversity in T. gondii may have been underestimated because most parasite strains in previous studies were collected from human patients and domestic animals in North America and Europe. To investigate the genetic diversity of T. gondii, we analysed parasite strains isolated from remote geographical regions by multilocus microsatellite sequencing and phylogenetic analysis. The genetic diversity indices, the molecular analysis of microsatellite genotypes and the constructed phylogram considered together suggest that the global T. gondii population is highly diversified and not characteristic of a clonal organism. The most parsimonious hypothesis is that T. gondii presents a complex population structure with a mix of clonal and sexual propagation as a function of the environmental conditions. The comparison between domestic strains data on one hand and wild strains data on the other hand is in favour of more frequent sexual recombinations in wild environment even though Toxoplasma subpopulation in human and domestic animals is largely clonal.International Journal for Parasitology 10/2004; 34(10):1185-96. · 3.64 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Outbreaks of toxoplasmosis are recognised infrequently. In March, 1995, a sudden increase of serologically diagnosed cases of acute toxoplasmosis was noted in the Greater Victoria area of British Columbia, Canada. Concurrently, but independently, seven cases of acute toxoplasma retinitis were diagnosed against a background of no cases in the previous 5 years. Cases were defined by serological testing, clinical presentation, and residence in Greater Victoria. A screening programme for women who were or had been pregnant was started. Geographical mapping of cases, and case-control studies of symptomatic cases and of women enrolled in the screening programme were done. 100 individuals aged 6 to 83 years met the definition for an acute, outbreak-related case. 94 resided in Greater Victoria and six had visited it; 19 had retinitis, 51 had lymphadenopathy, four others had symptoms consistent with toxoplasmosis, seven had other symptoms, 18 were symptom-free, and one would not provide information. 36 (0.9%) of 3812 screened pregnant and postnatal women were cases. Excess cases were not detected outside Greater Victoria and no conventional source of toxoplasmosis was implicated. Mapping studies of cases and of the screened women, and both case-control studies showed significant associations between acute infection and residence in the distribution system of one reservoir supplying water to Greater Victoria (ORs or RRs: 3.53, 3.05, 8.27, and 5.42, respectively). The epidemic curve appeared bimodal, with peaks in December, 1994, and March, 1995, that were preceded by increased rainfall and turbidity in the implicated reservoir. A municipal water system that uses unfiltered, chloraminated surface water was the likely source of this large community-wide outbreak of toxoplasmosis.The Lancet 08/1997; 350(9072):173-7. · 39.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Toxoplasma gondii affects a wide variety of hosts including threatened southern sea otters (Enhydra lutris nereis) which serve as sentinels for the detection of the parasite's transmission into marine ecosystems. Toxoplasmosis is a major cause of mortality and contributor to the slow rate of population recovery for southern sea otters in California. An updated seroprevalence analysis showed that 52% of 305 freshly dead, beachcast sea otters and 38% of 257 live sea otters sampled along the California coast from 1998 to 2004 were infected with T. gondii. Areas with high T. gondii exposure were predominantly sandy bays near urban centres with freshwater runoff. Genotypic characterisation of 15 new T. gondii isolates obtained from otters in 2004 identified only X alleles at B1 and SAG1. A total of 38/50 or 72% of all otter isolates so far examined have been infected with a Type X strain. Type X isolates were also obtained from a Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Molecular analysis using the C8 RAPD marker showed that the X isolates were more genetically heterogeneous than archetypal Type I, II and III genotypes of T. gondii. The origin and transmission of the Type X T. gondii genotype are not yet clear. Sea otters do not prey on known intermediate hosts for T. gondii and vertical transmission appears to play a minor role in maintaining infection in the populations. Therefore, the most likely source of infection is by infectious, environmentally resistant oocysts that are shed in the feces of felids and transported via freshwater runoff into the marine ecosystem. As nearshore predators, otters serve as sentinels of protozoal pathogen flow into the marine environment since they share the same environment and consume some of the same foods as humans. Investigation into the processes promoting T. gondii infections in sea otters will provide a better understanding of terrestrial parasite flow and the emergence of disease at the interface between wildlife, domestic animals and humans.International Journal for Parasitology 11/2005; 35(11-12):1155-68. · 3.64 Impact Factor
ORIGINAL ARTICLES AAEM
Infection with the parasitic protozoan Toxoplasma gon-
dii still represents an actual public health problem. Preva-
lence of the positive serologic reactions in humans varies
depending on geographical location, age, nutritional hab-
its, and keeping of hygienic standards [10, 34]. In Poland,
it is estimated at 50–60% [24, 28]. The T. gondii invasion
presents a particular hazard for pregnant women, with re-
spect to the possibility of foetus malformations, and for
THE OCCURRENCE OF TOXOPLASMA GONDII INFECTION IN PEOPLE
AND ANIMALS FROM RURAL ENVIRONMENT OF LUBLIN REGION
– ESTIMATE OF POTENTIAL ROLE OF WATER AS A SOURCE OF INFECTION
Jacek Sroka1, 2, Angelina Wójcik-Fatla1, Jolanta Szymańska3,
Jacek Dutkiewicz4, Violetta Zając1, Jacek Zwoliński1
1 Department of Occupational Biohazards, Institute of Agricultural Medicine, Lublin, Poland
2 Department of Parasitology, National Veterinary Research Institute, Puławy, Poland
3 Medical University of Lublin, Poland
4 Unit of Fibroproliferative Diseases, Institute of Agricultural Medicine, Lublin, Poland
Sroka J, Wójcik-Fatla A, Szymańska J, Dutkiewicz J, Zając V, Zwoliński J: The occur-
rence of Toxoplasma gondii infection in people and animals from rural environment of
Lublin region – estimate of potential role of water as a source of infection. Ann Agric
Environ Med 2010, 17, 125–132.
Abstract: A total of 254 humans and 489 domestic animals living on farms in the Lublin
province (eastern Poland) were examined for the presence of antibodies against Toxo-
plasma gondii using, respectively, the ELFA and direct agglutination tests. In parallel,
182 samples of potable water, mostly from shallow household wells, were taken on farms
and examined for the presence of T. gondii by microscopy and PCR. The frequency of se-
ropositive reactions in farm inhabitants (66.9%) was signifi cantly greater (p<0.01) com-
pared to the reference group of 39 healthy urban dwellers (41.0%). A highly signifi cant
positive correlation was found between the age of examined farm inhabitants and the rate
of positive reactions with Toxoplasma antigen (p<0.0001). Among domestic animals, the
greatest frequency of seropositive reactions to T. gondii occurred in cats (75.0%) and
dogs (53.6%), less frequent in cattle (33.8%) and hens (33.5%) and the least frequent in
pigs (17.9%) and ducks (21.2%). The presence of T. gondii was found in potable water
samples taken from water intakes on farms: in 12.6% of samples by microscopy, and in
22.5% of samples by PCR. Among 19 water samples taken from bathing places on the
territory of the Lublin province, 2 samples positive for T. gondii (10.5%) were found by
microscopic examination and confi rmed by PCR. The presence of live parasites in water
samples was demonstrated by the isolation of Toxoplasma gondii strains in mice. By
use of RFLP-PCR it was found that the majority of isolated Toxoplasma strains (78.0%)
belonged to clonal type I which is most virulent for humans and animals. Although no
statistically signifi cant relationship between the presence of T. gondii in water and oc-
currence of seropositive reactions in farm inhabitants and/or domestic animals could be
found, the above-mentioned data suggest a potential role of potable water in the spread of
toxoplasmosis in the rural environment.
Address for correspondence: Jacek Sroka, DVM, PhD, Department of Occupational
Biohazards, Institute of Agricultural Medicine, Jaczewskiego 2, 20-090 Lublin, Poland.
Key words: Toxoplasma gondii, epidemiology, serological examinations, farm inhabit-
ants, domestic animals, potable water, PCR.
Received: 3 November 2009
Accepted: 3 June 2010
Ann Agric Environ Med 2010, 17, 125–132
Sroka J, Wójcik-Fatla A, Szymańska J, Dutkiewicz J, Zając V, Zwoliński J
debilitated persons, in whom pathological changes may
occur in many organs or tissues. Recently, it was shown
that the invasion may cause distant effects in the form of
disturbances of sight and neural functions .
According to current views, humans contract toxoplas-
mosis mostly via the alimentary route, by eating raw meat
(mainly pork) containing cysts of T. gondii, or by con-
sumption other foods or water contaminated with oocysts
of the parasite [10, 11, 35]. However, at the assessment
of concrete clinical cases it is still not possible to deter-
mine which form of the parasite have caused the infection.
Hence, at the identifi cation of the source of infection in hu-
man clinical cases, it could be important to determine the
clonal type of parasite isolated.
The results obtained hitherto by our group and other au-
thors clearly indicate that toxoplasmosis may constitute a
serious epidemiological problem, particularly in the rural
environment where there are many cross routes of Toxo-
plasma transmission [10, 11, 28, 30]. Cats play a special
role in the spread of T. gondii, contaminating the environ-
ment with oocysts, the dispersive forms of this parasite.
Due to natural phenomena, oocysts are spread over a con-
siderable area which may also lead to the contamination of
surface and ground water. In this context, shallow house-
hold wells which are often the source of water supply in
rural areas of eastern Poland and are not covered by ex-
aminations for pathogenic protozoans, pose a potential risk
Hence, the objectives of this study were following:
• To determine the prevalence of seropositive reactions
to Toxoplasma gondii in farm inhabitants and domestic
• To determine the frequency of occurrence of Toxoplas-
ma gondii in drinking water from wells and water supply
systems, and in recreational waters in the rural areas of the
Lublin province, with respect to a potential health risk for
humans and domestic animals.
• To evaluate the role of water in the transmission of
Toxoplasma infection in the rural environment.
The present study is a continuation of the previous one
from 2006 , and concerns the next areas of the Lublin
MATERIALS AND METHODS
Examined farms. Studies of drinking water were con-
ducted on a total number of 164 farms in 35 villages located
in 12 districts of the Lublin province (eastern Poland), with
special consideration of the areas where high rates of se-
ropositive reactions were noted in earlier examinations of
humans and animals (districts of Włodawa, Parczew, and
Chełm). A total number of 182 samples of drinking water
was examined from the following intakes: 121 household
wells, 33 drilled wells, and 28 water supply system intakes.
Among household wells, 88 intakes were shallow wells
with a surrounding casing of concrete (depth up to 7 m) and
a manual winch, while the 33 remaining wells were 7–19 m
deep. The majority of wells were secured only by a provi-
sional wooden cover. 33 intakes were deeper (below 20 m)
drilled wells, where water was pumped by means of a man-
ual or electric pump. The samples from 28 intakes of the
water supply system were taken from taps inside houses.
Recreational waters. Water samples were taken from
19 bathing places on the territory of the Łęczna-Włodawa
Lakeland. The sites were located at 17 lakes (Piaseczno,
Rogóźno, Łukcze, Krasne, Białe, Białe Uścimowskie,
Maśluchowskie, Skomelno, Tomaszcze, Czarne, Zagłębocze,
Wytyckie, Miejskie, Bialskie, Czarne, Firlej, Jezioro), and
at 2 ponds (Wyklik, Siemień).
Questionnaire survey. A survey was conducted among
254 inhabitants of farms, aged 8–75 yrs, mean age 45.2 ±
20.4 yrs. The group constituted 154 women (60.6%) and
100 men (39.4%). The mean age of women was 44.4 ±
20.0 yrs and of men 46.5 ± 21.0 yrs.
The survey concerned: a) people living on a farm, eco-
nomic status, occurring diseases, nutritional habits; b) ani-
mals kept on farm, occurring diseases; c) water intakes,
ways of using water, frequency of its use.
Serologic studies of people and animals
living on farms
Serologic examination of people living on farms. A to-
tal of 254 farm inhabitants subjected to questionnaire study
and characterized above were examined. Reference group
consisted of 39 healthy persons living in the city of Lublin,
aged 23–59 yrs, mean age 37.7 ± 10.2 yrs. The reference
group constituted 30 women (mean age 37.6 ± 11.0 yrs)
and 9 men (mean age 38.1 ± 6.8 yrs).
Blood serum samples taken from the inhabitants of farms
and members of the reference group were examined by im-
munoenzymatic tests ELFA (Enzyme Linked Fluorescent
Assay) for the presence of IgG and IgM antibodies against
Toxoplasma gondii (Vidas Toxo IgG and Vidas Toxo IgM,
bioMérieux, Marcy l’Etoile, France). To perform the tests
by the ELFA technique, a Mini VIDAS device was used.
Serologic examination of animals kept on farms. A to-
tal of 489 domestic animals belonging to 11 species were
examined. The group constituted of: 74 cows, 67 pigs, 44
cats, 69 dogs, 14 horses, 7 goats, 5 rabbits and 209 speci-
mens of poultry, comprising 173 hens, 33 ducks, 2 turkeys
and 1 goose (Tab. 2).
Blood serum samples taken from the animals were ex-
amined by the direct agglutination test for the presence and
level of IgG antibodies against Toxoplasma gondii, using
commercial kit (Toxo-Screen DA, bioMérieux, Marcy
Toxoplasma gondii in people and animals
Study of water
Preparation of water samples. A total of 182 water
samples were taken into plastic cans with a volume of 50
litres. T. gondii isolation trials were performed based on the
method described by Isaac-Renton et al. . The samples
of water were fi ltered through cellulose fi lters with a pore di-
ameter of 0.45 μm. The fi lters were washed with phosphate
buffer (PBS) of pH 7.8, with the addition of 0.01% Tween
80, which was centrifuged for 4 min at 1,050 × g. The pellet
obtained was suspended in 20 ml of distilled water. Further
isolation was conducted by the fl otation method, with the
use of sugar solution of the following composition: 53 g
saccharose, 0.8 ml phenol, 100 ml distilled water (specifi c
gravity – 1.15 g). The pellet suspension was fi rst added to
30 ml of sugar solution and centrifuged for 4 min at 1,050 × g,
after which 25 ml of the liquid was sampled from the sur-
face, transferred to a new test-tube, and 75 ml PBS contain-
ing 0.01% Tween 80 was added. After fi nal centrifugation
for 10 min at 1,050 × g, the supernatant was removed, and
the sediment obtained was preserved for further studies.
Microscopic examination of water sediment. Sedi-
ment samples were suspended in a small amount of 0.9%
NaCl (in the proportion 1:2) and examined under a micro-
scope for the presence of T. gondii oocysts, using 200 ×
and 400 × magnifi cations. From each sample 5 prepara-
tions were made.
Bioassay on mice. Swiss-Webster females at the age
of 3 weeks were used. Before testing of each batch, circa
10% mice were randomly selected and their sera were ex-
amined by the direct agglutination test for the presence of
anti-Toxoplasma antibodies. All the examined mice proved
to be seronegative.
Samples of water sediments in which the presence of
T. gondii was stated by PCR test and/or microscopic exami-
nation were selected for testing. Each sample was suspend-
ed in the proportion 1:5 in 0.9% NaCl containing antibiot-
ics (1,000 U of penicillin and 100 μg of streptomycin per
1 ml). The suspension was given orally by stomach tube
to 3 mice in the volume of 0.5 ml per mouse. Mice were
observed daily, and in the case of appearance of clinical
symptoms (apathy, depression, diarrhea), they were killed
and sectioned. The specimens which died were also sub-
jected to section. During sections, samples of peritoneal
exudate, internal organs (liver, spleen, lungs) and brain
were taken for PCR test and histopathological examination,
and blood was drawn for serologic examination (direct ag-
glutination test). The remaining, healthy mice were killed
and sectioned 6 weeks after inoculation, and the samples of
peritoneal exudates and internal organs were examined as
described above. Everywhere, several blind passages were
performed, in which the next batch of mice were injected
intraperitoneally with the suspension of homogenized or-
gans from sectioned mice, after addition of antibiotics.
Polymerase Chain Reaction test (PCR). DNA isolation
from water sediment samples was performed using a com-
mercial kit (QIAmp DNA Mini Kit, Qiagen, Syngen Biotech,
Wrocław, Poland), according to manufacturer’s instruction.
Detection of T. gondii DNA based on amplifi cation of
35-fold-repetitive gene B1 fragment in 2 following nested
PCR reactions was performed with the method described
by Grigg and Boothroyd . The reaction mixture (50 μl)
contained: 1.5 U Tag DNA polymerase (Qiagen, Syngen
Biotech, Wrocław, Poland), 5 μl of the 10 × PCR Buffer
containing 1.5 mM MgCl2, 0.2 mM dNTPs (Polgen, Łódź,
Poland), 1 μl of each of the primers 10 mM: in the fi rst
reaction Pml/S1 (5’-TGTTCTGTCCTATCGCAACG)
and Pml/AS1 (5’-ACGGATGCAGTTCCTTTCTG), and
in the second nested-PCR reaction – Pml/S2 (5’-TCT-
TCCCAGACGTGGATTTC) and Pml/AS2 (5’-CTC-
GACAATACGCTGCTTGA) (Eurogentec, Seraing, Bel-
gium), nuclease-free water (Applied Biosystems, Warsaw,
Poland) and 5 μl matrix DNA.
Two-stage PCR reaction consisted of 30 and 20 cycles.
Each cycle included: the proper denaturation at 94°C for 30
sec, primers annealing at 60°C for 30 sec, and elongation at
72°C for 90 sec. The reaction products of stage I amplifi ca-
tions (5 μl) were used at stage II PCR. Additionally, at each
stage, the initial denaturation (2 min at 94°C), and the fi nal
elongation (2 min at 72°C) were performed. The 531 bp-long
products of the second amplifi cation were detected in 2%
agarose gel (Prona, Basica L.E.) after staining with a water
solution of ethidium bromide. The Toxoplasma gondii strains:
RH (type I), ME49 (type II), and C56 (type III) were used as
positive control, while nuclease-free water (Applied Biosys-
tems, Warsaw, Poland) was used as a negative control.
To identify the clonal type (I or II/III) of the isolated
T. gondii strains, RFLP PCR was performed. The PCR am-
plifi cation products were treated with restriction enzymes:
Eco 721 and Xhol (Fermentas, Vilnius, Lithuania), and the
reaction products detected in 2% agarose gel.
DNA sequencing was performed at the DNA Sequencing
and Oligonucleotides Laboratory (Institute of Biochemis-
try and Biophysics, Polish Academy of Sciences, Warsaw,
Poland) and at the Institute of Agricultural Medicine in
Lublin, Poland. The results were compared to published
sequences in the GenBank database using the BLAST
server at the National Center for Biotechnology Informa-
tion (Bethesda, Maryland, USA).
Statistical analysis. The data was analysed with chi-
square (χ2) test and t-Student’s test using Statistica 8.0
package (Statsoft Inc., Tulsa, OK, USA).
All the examined 254 people lived on farms and had
everyday contact with animals, and all adults reported
Sroka J, Wójcik-Fatla A, Szymańska J, Dutkiewicz J, Zając V, Zwoliński J
the agricultural profession. 10 individuals had in the past
symptoms suggesting the nodal form of toxoplasmosis
(one person was treated for this disease), and 7 women
had disorders during pregnancy. All 17 persons had anti-
Toxoplasma antibodies of IgG class. The inhabitants of 73
out of 164 farms reported drinking unboiled water, while
inhabitants of 54 farms reported consumption of raw meat.
The inhabitants of 30 farms reported biting by ticks in the
The majority of examined farms were small, with free-
run rearing of poultry and seasonal grazing of cattle and
goats near farm buildings. On these farms, the animals were
kept in improper zoohygienic conditions and fed with self-
produced fodder. It was stated that cats kept on farms had
free access to human residential areas, as well as to animal
houses and fodder stores. In 14 farms there occurred in the
past cases of disease in animals, associated with abortions
and/or deaths of cows, calves, piglets, horses and cats. On
the majority of farms, the household wells were used not
only for consumption by people and animals, but also for
other purposes, such as watering of gardens.
Serologic response of people and animals
to Toxoplasma gondii
Serologic response of people. 170 out of 254 examined
farm inhabitants (66.9%) showed the presence of anti-
Toxoplasma antibodies of IgG class, while only 5 (2.0%)
showed the presence of antibodies of IgM class (Tab. 1).
Most of the IgG-dependent positive results were weakly
or mediocre positive (50.6% and 47.6%, respectively),
while only a small proportion (1.8%) was strongly posi-
tive (Tab. 1). Prevalence of seropositive results in men and
women was similar (66.0% vs 67.5%). Rates of positive
reactions in women living on farms and total farm inhabit-
ants were signifi cantly greater compared to the reference
group (respectively, 40.0% and 41.0%, p<0.01). The dif-
ference was not signifi cant in the case of men from the ref-
erence group (44.4%, p=0.19).
A highly signifi cant positive correlation was found be-
tween the age of examined farm inhabitants and the rate of
positive reactions with Toxoplasma antigen (Fig. 1). The
mean age of seropositive farm inhabitants was signifi cantly
greater compared to that of seronegative ones: in the case
of women it was 50.8 vs 30.9 yrs (p<0.0001), in the case of
men – 54.5 vs 31.0 yrs (p<0.0001), and in the case of the
total examined – 52.3 vs 30.9 yrs (p<0.0001).
Moderate higher rates of seropositive results occurred
among the people on these farms, where cases of disease
in animals (associated with deaths and/or abortions) were
reported in the past, but difference was not signifi cant
(69.3% vs 66.9%, p=0.83).
No signifi cant difference was found between the rates of
positive reactions to Toxoplasma in the inhabitants of these
farms in which T. gondii DNA was found in water, and the
remainder (57.4% vs 69.2%, p>0.05). Similarly, the rates
of positive reactions to Toxoplasma in the group of farm
inhabitants using wells compared to those using water sup-
ply system did not show a signifi cant difference (66.0%
vs 67.8%, p>0.05). In farm inhabitants reporting consump-
tion of unboiled water the rate of positive anti-Toxoplasma
reactions was greater compared to those drinking boiled
water, but the difference did not attain signifi cance level
(67.9% vs 61.3%, p>0.05).
Serologic response of animals. A total of 177 out of
489 examined animals (36.2%) showed seropositive re-
actions to Toxoplasma gondii (Tab. 2). The highest rates
Table 1. Prevalence of seropositive reactions to Toxoplasma gondii in
farm inhabitants vs reference group assessed by ELFA test.
Group IgG-dependent reactions
number of positive (percent)
IU – International Units; N – number of examined persons; * signifi cantly
greater compared to reference group (p<0.01).
0–15 16–3031–45 46–6061and>
Figure 1. Correlation between age of examined farm inhabitants and
prevalence of positive serologic reactions to Toxoplasma gondii.
Toxoplasma gondii in people and animals
of positive results were noted in cats (75.0%) and goats
(71.4%). The lower response rates were found in dogs
(53.6%), cows (33.8%), hens (33.5%), ducks (21.2%), and
pigs (17.9%). No positive results were noted in 14 horses,
5 rabbits, 2 turkeys and 1 goose. Most of the animals re-
acted in high titers (13.9%), less in mediocre (9.2%) and
low titers (13.1%) (Tab. 2).
No signifi cant difference was found between the fre-
quencies of positive results in animals on farms where
T. gondii DNA was detected in water by PCR, compared to
farms with negative results of water examination (38.1%
vs 35.7%, p>0.05). Similarly, no signifi cant difference was
noted between the frequencies of positive results in ani-
mals on farms using well water, compared to those using
water supply system (35.0% vs 39.3%, p>0.05).
Studies of water
Microscopic examination. Among a total number of
182 drinking water samples, in 23 samples (12.6%) the
presence of individual oocysts of the size 9–10 × 12–13 μm
was noted, which were considered as Toxoplasma gondii
oocysts. These oocysts possessed a double wall and vari-
able internal structure: from homogenous, granular proto-
plasm to visible outlines of 2 sporocysts. 21 positive wa-
ter samples came from shallow wells, 2 from deep drilled
wells and none from intakes of the water supply system
(Tab. 3). The prevalence of Toxoplasma in shallow wells
was signifi cantly greater than in intakes of the water supply
PCR test. Among 182 water samples examined, the pres-
ence of T. gondii DNA was detected in 41 samples (22.5%),
including: 31 samples from shallow wells (25.6%), 5 sam-
ples from deep wells (15.2%), and 5 samples from intakes
of the water supply system (17.9%). No signifi cant differ-
ences were found between the frequencies of T. gondii in
water from different sources.
The presence of T. gondii DNA in the PCR-positive sam-
ples was confi rmed by sequencing. Using RFLP-PCR, the
clonal types of 41 positive samples were determined. Most
of the T. gondii DNA isolates (32 samples) were classifi ed
into the virulent for mice type I, and 3 isolates into type
II/III. The remaining 6 isolates were classifi ed as “atypi-
cal” (Tab. 4).
Among 19 water samples taken from bathing places on
the territory of the Lublin province, 2 samples positive for
T. gondii (10.5%) were found by microscopic examination,
and confi rmed by PCR. Using the RFLP-PCR analysis,
Table 2. Prevalence of seropositive reactions to Toxoplasma gondii in
domestic animals assessed by direct agglutination test.
Animal speciesTiters of positive reactions
number of positive (percent)
Pigs 673 (4.5) 4 (6.0)5 (7.4) 12 (17.9)
Cows 74 22 (29.7)0 3 (4.1) 25 (33.8)
Cats 440 3 (6.8)30 (68.2) 33 (75.0)
Dogs 69 16 (23.2)12 (17.4) 9 (13.0) 37 (53.6)
Goats 71 (14.3)0 4 (57.1)5 (71.4)
Hens 17317 (9.8)26 (15.0)15 (8.7) 58 (33.5)
Ducks 335 (15.2)02 (6.0) 7 (21.2)
Total 48964 (13.1) 45 (9.2)68 (13.9) 177 (36.2)
N – number of examined animals.
Table 3. Occurrence of Toxoplasma gondii in water samples taken from
water intakes located on farms.
Positive samples: number (percent)
Deep wells Water
N – number of examined samples.
* Only 14 samples positive in PCR test were examined.
** Result of PCR testing of tissues and/or peritoneal exudate of mice in-
oculated with sediments of positive water samples. By microscopy, toxo-
plasmas were found in mice tissues and/or peritoneal exudate only in 5
out of 14 water samples (35.7%).
Table 4. Prevalence of clonal types of T. gondii (determined by RFLP-
PCR) in water samples from water intakes located on farms, depending
on type of intake.
of T. gondii
Water intake: number of positive samples (percent)
N – number of examined samples.
Sroka J, Wójcik-Fatla A, Szymańska J, Dutkiewicz J, Zając V, Zwoliński J
it was determined that one T. gondii isolate (from Białe
Lake) pertained to the clonal type II/III while the other
(from Maśluchowskie Lake) pertained to type I.
Bioassay on mice. Toxoplasmas (tachyzoites or brady-
zoites) were found microscopically in tissues and/or perito-
neal exudate in mice inoculated with 5 out of 14 sediments
of water samples found earlier to be positive in PCR test
for T. gondii. All microscopy fi ndings were confi rmed by
PCR test, which was also positive in mice inoculated with
further 4 sediments, so altogether, bioassay on mice proved
to be positive in 9 out of 14 PCR-positive water samples
(64.3%). The positive result of PCR test for mice tissues
and/or peritoneal exudates was obtained in 8 samples only
in the fi rst passage, and in 1 sample also in the second pas-
sage. All results for the third passage were negative. The
results of serological examination were positive only in
mice inoculated with 3 out of 14 water samples, in titers
ranging from 64–540.
The strains of T. gondii isolated from 2 water sediment
samples pertained to clonal type I (virulent for mice) and
an atypical one, causing quick deaths of mice after inocu-
lation. The results of serological tests in these mice were
negative or doubtful. The mice inoculated with water sedi-
ments from which T. gondii of II/III type was isolated, sur-
vived in good condition 6 weeks until the section, and the
presence of T. gondii was confi rmed by PCR and/or micro-
scopy only in brain tissue.
The results of this study show that people living on farms
are infected with Toxoplasma gondii signifi cantly more of-
ten than urban dwellers, and that the infection rate in farm
inhabitants increases signifi cantly with age. Similar rela-
tionships were demonstrated by Studeničová et al. ,
Nash et al. , Bibic et al.  and Sroka . The lack of
these correlations were observed by Studeničová et al. 
and Sousa et al. . Other results of the present work in-
dicate that one of the reasons for the common occurrence of
Toxoplasma infection among farm inhabitants may be the
close contact with domestic animals (and with their tissues)
which could be infected with T. gondii at high rates.
Because of the common meat consumption, slaughter
animals are regarded as an important source of T. gondii
infection in Europe . The results obtained in this study
confi rmed the signifi cant role of pigs in the epidemiology
of toxoplasmosis (infection rate amounted to 17.9%). The
percentage of positive results detected in pigs is higher
than previously obtained in the same areas (10.5%) 
and in Wielkopolska region (Poland) . The high rates
of seropositive results obtained in this study among cats
and goats populations (respectively, 75.0% and 71.4%)
showed that these species may also play an important role
in the transmission of this parasite in the rural environment
of Lublin region. As poultry meat is usually frozen, it does
not constitute a frequent source of infection for humans.
Similarly, cattle, despite the high seropositivity noted, are
regarded as a rare source of infection .
Considering the potential sources of toxoplasmic infec-
tion both for the people and animals living on farms, spe-
cial attention must be paid to potable water. On the world
scale, a number of waterborne cases of toxoplasmosis in
humans have been described [2, 3, 6, 16]. However, a rela-
tively small number of studies have been devoted to the
transmission of Toxoplasma by water, largely because of
methodological diffi culties. The application of PCR, as a
method more sensitive compared to microscopy, increased
the effectiveness of detection of parasites in water [20, 21,
36]. Nevertheless, most researchers are of opinion that
people contract toxoplasmosis mainly by eating raw or
undercooked meat containing cysts of Toxoplasma, or by
consumption of products contaminated with faeces of sick
cats, containing oocysts [10, 34, 35].
In the earlier work  we found a signifi cant correla-
tion between the consumption of unboiled well water and
the presence of specifi c anti-Toxoplasma antibodies in farm
inhabitants, particularly on farms of poor hygienic state,
including shallow wells. Similarly, other authors analyzing
cases of T. gondii infection in various populations, also not-
ed a signifi cant correlation between the drinking of unboiled
water and occurrence of Toxoplasma invasion in examined
people [9, 12, 22, 24]. The demonstration in the present
work of the considerable frequency of T. gondii in the sam-
ples of potable water taken on farms (12.6% by microscopy
and 22.5% by PCR) suggests that water may play an im-
portant role in spreading of toxoplasmosis in the rural en-
vironment. These fi ndings were confi rmed by the isolation
from water samples of the virulent T. gondii strains in mice.
However, in this work we were not able to fi nd a signifi cant
relationship between the presence of T. gondii in water and
the occurrence of seropositive reactions in farm inhabitants
and domestic animals. This suggests that for the majority
of examined people and animals the sources of Toxoplasma
invasion could probably be different from potable water.
Depending on variability in genetic material and viru-
lence for mice, 3 main clonal types are distinguished among
the T. gondii strains: type I (virulent for mice), and types II
and III (avirulent for mice) [14, 18]. Strains of type II are re-
sponsible mainly for invasions in animals, whereas strains
isolated from human clinical cases often belong to type I or
type III [5, 17, 18]. More recently, also so called “atypical”
strains have been distinguished [1, 13, 15] which could be
found as dominant, mostly in studies of the environment
[7, 11]. In the present study, genotyping of the T. gondii
DNA isolates based on the detection of the differences in
the fragment of B1 gene, allowed for the differentiation of
type I from type II and/or III . The majority of the iso-
lates from water samples (78.0%) were classifi ed into type
I, while strains II/III and atypical were less common (7.3%
and 14.7%, respectively). The prevalence of virulent type
I in water samples could be recognized as hazardous from
Toxoplasma gondii in people and animals
the epidemiological point of view. Nevertheless, for the full
characteristics of the isolated T. gondii strains an analysis
based on the greater number of markers is recommended,
for example: B1, SAG1-SAG3, GRA6, L363. Genotyping
of Toxoplasma strains is regarded by leading researchers as
a valuable part of epidemiological investigation and an im-
portant marker of health hazard for the people .
In spite of the lack of a signifi cant relationship in this
study between the presence of T. gondii in water and oc-
currence of seropositive reactions in farm inhabitants and
domestic animals, the role of potable water in the spread-
ing of toxoplasmosis in rural areas cannot be excluded.
On most of the farms, simultaneous infections occurred in
several farm inhabitants and/or in the majority of domes-
tic animals, which indicates the existence of the infection
sources, including water intakes.
To summarize, the results of the present study and those
by other authors justify the necessity to implement the
sanitary monitoring of rural household wells and to sup-
plement the scope of routine examinations of water from
water supply systems by studies for parasitic protozoans.
The implementation of preventive measures on farms at
risk would also be important.
1. People living on farms showed a signifi cantly greater
serological response to Toxoplasma gondii than healthy ur-
ban dwellers constituting the reference group.
2. Among examined species of animals the greatest fre-
quency of seropositive reactions to T. gondii occurred in
cats, goats and dogs, less so in cattle, poultry and pigs.
3. The presence of T. gondii was found in nearly one
quarter of potable water samples taken from water intakes
on farms, and the majority of isolated Toxoplasma strains
belonged to clonal type I, which is most virulent for hu-
mans and animals. The lack of a signifi cant relationship
between the presence of T. gondii in water and the occur-
rence of seropositive reactions in farm inhabitants and/or
domestic animals suggest that apart from potable water,
other important sources of infection also exist in the sur-
roundings of examined people and animals.
This study was supported by the Polish Ministry of Science and
Higher Education (Grant No. N 404 071 31/3214).
1. Ajzenberg D, Banuls AL, Su C, Dumetre A, Demar M, Carme B,
Dardé ML: Genetic diversity, clonality and sexuality in Toxoplasma gon-
dii. Int J Parasitol 2004, 34, 1185–1196.
2. Bahia-Oliveira LMG, Jones JL, Azevedo-Silva J, Alves CCF, Oré-
fi ce F, Addiss DG: Highly endemic, waterborne toxoplasmosis in north
Rio de Janeiro state, Brazil. Emerg Infect Dis 2003, 9, 55–62.
3. Benenson MW, Takafuji ET, Lemon SM, Greenup RL, Sulzer AJ:
Oocyst-transmitted toxoplasmosis associated with ingestion of contami-
nated water. N Engl J Med 1982, 307, 666–669.
4. Bobic B, Nikolic A, Klun I, Vujanic M, Djurkovic-Djakovic O:
Undercooked meat consumption remains the major risk factor for Toxo-
plasma infection in Serbia. Parassitologia 2007, 49, 227–230.
5. Boothroyd JC, Grigg ME: Population biology of Toxoplasma gon-
dii and its relevance to human infection: do different strains cause differ-
ent disease? Curr Opin Microbiol 2002, 5, 438–442.
6. Bowie WR, King AS, Werker DH, Isaac-Renton JL, Bell A, Eng SB,
Marion SA: Outbreak of toxoplasmosis associated with municipal drinking
water. The BC Toxoplasma Investigation Team. Lancet 1997, 350, 173–177.
7. Conrad PA, Miller MA, Kreuder C, James ER, Mazet J, Dabritz
H, Jessup DA, Gulland F, Grigg ME: Transmission of Toxoplasma: clues
from the study of sea otters as sentinels of Toxoplasma gondii fl ow into
the marine environment. Int J Parasitol 2005, 35, 1155–1168.
8. Cook AJC, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum
PA, Foulon W, Semprini AE, Dunn DT: Sources of toxoplasma infection
in pregnant women: European multi centre case -control study. BMJ 2000,
9. Dawson D: Foodborne protozoan parasites. Int Food Microbiol
2005, 103, 207–227.
10. Dubey JP, Beattie CP: Toxoplasmosis of Animals and Man. CRC
Press Inc., Boca Raton 1988.
11. Dubey JP, Graham DH, De Young RW, Dahl E, Eberhard ML, Nace
EK, Won K, Bishop H, Punksody G, Streekmar C, Vianna MC, Shen SK,
Kwok OC, Summers JA, Demarais S, Hill DE, Chirukandoth S, Dubey
JP: Biology and epidemiology of Toxoplasma gondii in man and animals.
Anim Health Res Rev 2005, 6, 41–61.
12. Ertug S, Okyay P, Turkmen M, Yukse H: Seroprevalence and risk
factors for Toxoplasma infection among pregnant women in Aydin prov-
ince, Turkey. BMC Public Health 2005, 5, 66.
13. Ferreira AM, Vitor RWA, Carneiro ACAV, Brandão GP, Melo MN:
Genetic variability of Brazilian Toxoplasma gondii strains detected by
random amplifi ed polymorphic DNA-polymerase chain reaction (RAPD-
PCR) and simple sequence repeat anchored PCR (SSR-PCR). Infect
Genet Evol 2004, 4, 131–142.
14. Grigg ME, Boothroyd JC: Rapid identifi cation of virulent type I
strains of the protozoan pathogen Toxoplasma gondii by PCR restriction
fragment length polymorphism analysis at the B1 gene. J Clin Microbiol
2001, 39, 398–400.
15. Grigg ME, Suzuki Y: Sexual recombination and clonal evolution
of virulence in Toxoplasma. Microbes Infect 2003, 5, 685–690.
16. Hall SM, Pandit A, Golwilkar A, Williams TS: How do Jains get
Toxoplasma infection? Lancet 1999, 354, 486–487.
17. Howe DK, Honoré S, Derouin F, Sibley LD: Determination of
genotypes of Toxoplasma gondii strains isolated from patients with toxo-
plasmosis. J Clin Microbiol 1997, 35, 1411–1414.
18. Howe DK, Sibley LD: Toxoplasma gondii comprises three clonal
lineages: correlation of parasite genotype with human disease. J Infect
Dis 1995, 172, 1561–1566.
19. Isaac-Renton J, Bowie WR, King A, Irwin GS, Ong CS, Fung CP,
Shokeir MO, Dubey JP: Detection of Toxoplasma gondii oocysts in drink-
ing water. Appl Environ Microbiol 1998, 64, 2278–2280.
20. Kellogg J, McDevitt S, McDevitt JJ: Development of a PCR-enzyme
immunoassay oligoprobe detection method for Toxoplasma gondii oocysts,
incorporating PCR controls. Appl Environ Microbiol 2003, 69, 5819–5825.
21. Kourenti C, Heckeroth A, Tenter A, Karanis P: Development and
application of different methods for the detection of Toxoplasma gondii in
water. Appl Environ Microbiol 2003, 69, 102–106.
22. Lopez-Castillo CA, Diaz-Ramirez J, Gomez-Marin JE: Risk fac-
tors for Toxoplasma gondii infection in pregnant women in Armenia, Co-
lombia. Rev Salud Publica 2005, 7, 180–190.
23. Nash JQ, Chissel S, Jones J, Warburton F, Verlander NQ: Risk
factors for toxoplasmosis in pregnant women in Kent, United Kingdom.
Epidemiol Infect 2005, 133, 475–483.
24. Paul M: Potencjalne źródła zarażenia Toxoplasma gondii w przy-
padkach badanych w krótkim czasie po zarażeniu. Przegl Epidemiol
1998, 52, 447–454.
25. Pawłowski ZS: Toksoplazmoza w Wielkopolsce w latach 1990–
2000. Przegl Epidemiol 2002, 56, 409–417.
26. Petersen E, Pollak A, Reiter-Owona I: Recent trends in research on
congenital toxoplasmosis. Int J Parasitol 2001, 31, 115–144.
Sroka J, Wójcik-Fatla A, Szymańska J, Dutkiewicz J, Zając V, Zwoliński J
27. Sousa W, Coutinho S, Lopes C, Dos Santos C, Neves N, Crus A:
Epidemiological aspects of toxoplasmosis in school children residing in
localities in the urban or rural characteristics within the city of Rio de
Janeiro, Brazil. Mem Inst Oswaldo Cruz 1987, 82, 475–482.
28. Sroka J: Badania nad występowaniem Toxoplasma gondii
u zwierząt hodowlanych i dzikich z terenu województwa lubelskiego
w aspekcie zagrożenia zdrowia ludności wiejskiej. Doctoral Thesis. The
Faculty of Veterinary Medicine, Agricultural University of Lublin, Lublin
29. Sroka J: Seroepidemiology of toxoplasmosis in the Lublin region.
Ann Agric Environ Med 2001, 8, 25–31.
30. Sroka J, Wójcik-Fatla A, Dutkiewicz J: Occurrence of Toxoplasma
gondii in the water from wells located on farms. Ann Agric Environ Med
2006, 13, 169–175.
31. Sroka J, Zwoliński J, Dutkiewicz J: Seroprevalence of Toxoplasma
gondii in farm and wild animals from the area of Lublin province. Bull Vet
Pulawy 2007, 51, 535–540.
32. Studeničová C, Benčaiová G, Holková R: Seroprevalence of Toxo-
plasma gondii antibodies in a healthy population from Slovakia. Eur J
Intern Med 2006, 17, 470–473.
33. Studeničová C, Ondriska F, Holková R: Seroprevalence of Toxo-
plasma gondii among pregnant women in Slovakia. Epidemiol Mikrobiol
Imunol 2008, 57, 8–13.
34. Su C, Evans D, Cole RH, Kissinger JC, Ajioka JW, Sibley LD:
Recent expansion of Toxoplasma through enhanced oral transmission.
Science 2003, 299, 414–416.
35. Tenter AM, Heckeroth AR, Weiss LM: Toxoplasma gondii: from
animals to humans. Int J Parasitol 2000, 30, 1217–1258.
36. Villena I, Aubert D, Gomis P, Ferté H, Inglard JC, Denis-Bisiaux
H, Dondon JM, Pisano E, Ortis N, Pinon JM: Evaluation of strategy for
Toxoplasma gondii oocyst detection in water. Appl Environ Microbiol
2004, 70, 4035–4099.
37. Villena I, Marle M, Dardé ML, Pinon JM, Aubert D: Toxoplasma
strain type and human disease: Risk of bias during parasite isolation?
Trends Parasitol 2004, 20, 160–162.