Short communication

Prevalence of Toxoplasma gondii infection in Belgian house cats

Stephane De Craeye a, Aurelie Francart b, Julie Chabauty a, Veerle De Vriendt c,Steven Van Gucht b, Ingrid Leroux b, Erik Jongert a,*

a Laboratory for Toxoplasmosis, Pasteur Institute of Brussels, Scientific Institute of Public Health, Brussels, Belgiumb Rabies Laboratory, Pasteur Institute of Brussels, Scientific Institute of Public Health, Brussels, Belgium

c Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

Received 25 January 2008; received in revised form 27 May 2008; accepted 10 July 2008

Abstract

Five hundred and sixty seven sera of healthy house cats aged 3 months to 7 years, were examined for the presence of anti-

toxoplasma antibodies by indirect immunofluorescence assay and compared to SAG1 and TLA enzyme linked immunosorbent

assays as alternative test. Twenty-five percent of cats tested positive for IgG and/or IgM. Seroprevalence increased with age from

2% below 12 months of age up to 44% at age 7. Sensitivities of SAG1 and TLA ELISA were 84.1% and 88.6%, respectively. Peak

levels in seroprevalence were correlated to increased IgG titers in TLA ELISA. Our results suggest that T. gondii infections are

common in house cats and that there is a high chance for a negative cat to seroconvert in its second life-year.

Crown Copyright # 2008 Published by Elsevier B.V. All rights reserved.

Keywords: Toxoplasma gondii; Zoonosis; Seroprevalence; Transmission; ELISA; Indirect immunofluorescence assay; SAG1; TLA; Titer;

Antibody

www.elsevier.com/locate/vetpar

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Veterinary Parasitology 157 (2008) 128–132

1. Introduction

Toxoplasma gondii is an obligate intracellular

parasite and infects almost all warm-blooded animals

and humans (Tenter et al., 2000). Cats get infected by

feeding on infected meat (prey or treat), or by ingestion

of sporulated oocysts, resulting in an enteroepithelial

sexual cycle that leads to the shedding of oocysts in the

environment (Dubey, 1986). Those oocysts rapidly

sporulate and stay infectious for more than a year

(Dubey, 1998). Toxoplasma may lead to serious

morbidity and mortality in seronegative pregnant

women and immunocompromised patients (Montoya

and Liesenfeld, 2004), and to chorioretinitis in

otherwise healthy individuals (Gilbert et al., 2006). A

* Corresponding author. Tel.: +32 3 373 33 77; fax: +32 2 373 32 81.

E-mail address: ejongert@pasteur.be (E. Jongert).

0304-4017/$ – see front matter. Crown Copyright # 2008 Published by E

doi:10.1016/j.vetpar.2008.07.001

recent European multi-centre study has indicated that

infected meat is considered as the main risk for

congenital toxoplasmosis (Cook et al., 2000). However,

oocyst contamination plays an important role in

transmission of this parasite as strict vegetarians

become infected with Toxoplasma (Hall et al., 1999;

Roghmann et al., 1999). Toxoplasma seroprevalence in

humans has been associated with living in close

proximity to Toxoplasma seropositive cats: children

who had cats were more likely to be infected than

children who had no cats (Pereira et al., 1992), and

toxoplasma seropositivity in adults was associated with

living in close proximity to seropositive cats (Sukthana

et al., 2003). In Belgium, about 50% of the population is

infected with T. gondii and congenital toxoplasmosis

occurs in 9/10,000 pregnancies (Breugelmans et al.,

2004). The cat population in Belgium is quite dense and

about 26% of households has at least one cat (DGS,

2007).

lsevier B.V. All rights reserved.

S. De Craeye et al. / Veterinary Parasitology 157 (2008) 128–132 129

The aim of this study was to determine the

prevalence of toxoplasma infection in house cats

ranging from 3 months to 7 years of age. Different

serological tests were used to evaluate antibody

responses against T. gondii and to evaluate whether

they could match the IIFA reference test for cats.

2. Materials and methods

2.1. Sera

Serum from healthy house cats aged 3 months to

7 years was obtained from the National Reference

Laboratory for Rabies at the Pasteur Institute of

Brussels, Scientific Institute for Public Health. These

sera were originally submitted for titration of rabies

antibodies in a virus-neutralisation assay, which

requires the samples to be complement-inactivated

at 56 8C for 30 min. This test has to be performed

in accordance with the European pet travel scheme, to

allow the owners to enter certain rabies-free

countries with their pets. All sera were taken by

private veterinary practitioners between 2004 and

2006, prior to the travel of the animal outside the

Belgian territory, and were provided with birth date

of the cat and date of serum sampling. Samples

were stored at �20 8C. All animals were healthy at

time of sampling. Per age group, 49–90 samples were

selected.

2.2. Indirect immunofluorescence assay (IIFA)

Fifty microliter of each serum diluted 1/50 in

phosphate-buffered saline (PBS) was applied on slides

coated with formalin-treated tachyzoites from the RH

strain (Toxo-Spot IF, Bio-Merieux, France) and

incubated for 30 min at 37 8C. Slides were washed

with PBS and incubated for 30 min at 37 8C with 30 ml

of fluorescein isothiocyanate conjugated goat-anti-cat

IgM (m)- or goat-anti-cat IgG (H+L) (KPL, Gaithers-

burg, MA) diluted 1/20 and 1/40 in PBS, respectively,

and stained with Evans Blue as counter dye. After

washing and drying, the slides were read with a

fluorescence microscope (Carl Zeiss). The cut-off

read-out of the fluorescence test was established at

a dilution of 1/40 with Toxoplasma negative and

positive feline reference sera from our laboratory,

according to Toxo-Spot IF guidelines. Under ultra-

violet light, the surface of the tachyzoite is fluorescent

green with positive serum, while for seronegative

samples the tachyzoite will show a red background

stain. The specificity and sensitivity of the Toxo-

Spot IF test for IgG are 98.44% and 95.08%,

respectively, while for IgM it is not detailed by the

manufacturer.

2.3. Parasites and total lysate antigen

Total lysate antigen (TLA) was prepared from RH

tachyzoites as described previously (Jongert et al.,

2007).

2.4. Enzyme linked immunosorbent assays (ELISA)

SAG1 coated ELISA plates (Meddens Diagnostics

BV, The Netherlands) were incubated with cat sera

diluted 1/50 in PBS with 10% foetal calf serum (FCS).

SAG1-specific IgG was detected using an horseradish

peroxidase (HRP) conjugated anti-cat IgG diluted 1/

10.000 in 10% FCS in PBS (AbD Serotec, Oxford, UK).

Colorimetric reaction was done with 3, 30, 5, 50-tetramethylbenzidine liquid substrate system (Sigma,

St. Louis, MO) and stopped with 0.1N H2SO4. Plates

were read at 450 nm in a spectrophotometer (Bio-Rad,

Hercules, CA). Sera were considered positive if the

optical density (OD450) exceeded the cut-off value (=

mean OD450 + 3� Standard Deviation OD450) from

three reference Toxoplasma negative cat sera at dilution

1/50. Determination of the IgG endpoint titer against

TLA was performed as described previously with some

minor modifications (Jongert et al., 2007). Briefly, 96-

well ELISA Maxisorp plates (Nunc, Roskilde, Den-

mark) were coated with 10 mg/ml TLA overnight at

4 8C. After blocking with 10% FCS in PBS for 2 h at

37 8C, IIFA positive cat sera were applied in twofold

dilutions starting at a dilution of 1/50 and incubated

overnight at 4 8C. ELISA plates were further treated as

described above. Endpoint titers were defined as the

highest dilution where the optical density (OD450)

exceeded the cut-off value calculated with the OD450

from two reference Toxoplasma negative cat sera at the

same dilution.

2.5. Statistical analysis

Logistic regression was used to investigate the

correlation between Toxoplasma seroprevalence and

age using SPSS 13 software (SPSS, Chicago, IL), and

the correlation between antibody endpoint titer

and seroprevalence per age group was investigated

by Chi-square test for trend. Titer distributions were

evaluated for normality with Kolmogorov–Smirnov

test.

S. De Craeye et al. / Veterinary Parasitology 157 (2008) 128–132130

3. Results

3.1. Toxoplasma seroprevalence in Belgian house

cats

Twenty-five percent of the 567 selected sera were

positive for IgG and/or IgM in IIFA reference test

(Table 1). Toxoplasma-specific IgM antibodies

occurred in 2.5–7% of cats aged 12 months to 6 years.

Only one cat in the age group of 2 years showed IgM but

no IgG antibodies. An important amount of T. gondii-

specific IgM antibodies was observed in the age group

of 7 years. Seroprevalence increased with age: merely

2% below 12 months, 13–26% at 12 months to 5 years,

and 40–44% at 6–7 years. Logistic regression on

continuous age data showed that the increase in

seroprevalence was positively correlated with increase

in age (b = �1.115, S.E. = 0.097, P = 0.0001). Peak

levels in seroprevalence were observed at 1 year of age

(17.2% increase) and, remarkably, also in the 6-year

age group (14.4% increase), while in age groups of 2–5

years this remained relatively unchanged. The average

increase in seroprevalence per life-year was

6.25 � 2.53% S.E.M. In order to evaluate whether

serologic screening by ELISA could match the IIFA

reference test for cats, IIFA Toxoplasma-positive sera

were evaluated against SAG1 and TLA in ELISA.

Overall, ELISA could confirm the presence of anti-

SAG1 or anti-TLA IgG in 84.1% and 88.6% of IIFA

positive samples, respectively, with variable sensitivity

per age group (Table 1).

Table 1

Evaluation of Toxoplasma-specific antibody response per age-group

Cat age (years) Samples IIFA

IgM+a IgG+a Seropositive

0 49 0 1 2%

1 78 3 15 19.2%

2 90 3d 12 13.3%

3 78 2 20 25.6%

4 74 5 19 25.7%

5 68 3 18 26.5%

6 71 5 29 40.8%

7 59 10 26 44.1%

Total 567 31 140 24.9%e

a Number of Toxoplasma-positive cats identified by IIFA.b Percentage of IIFA tested positive cat sera that is positive in ELISA.c Mean IgG endpoint titer against TLA per age group, with S.E.M. and rand One cat serum was only IgM positive.e Mean seroprevalence on total cat number.f Sensitivity of ELISA test compared to the total number of IIFA positivg Mean IgG endpoint titer against TLA of all IIFA positive samples. n.d

3.2. Increased IgG endpoint titers coincide with

increases in seroprevalence

In order to evaluate whether the peaks in seropre-

valence in the age groups of 12–23 months and 6 years

could have been indicative of a recent infection, anti-

Toxoplasma antibody titers from all IIFA positive cat

sera were tested in TLA ELISA (Table 1). Average titers

per age group showed higher IgG titers at the ages 12–

23 months and 6 years. Chi-square test for trend showed

that there was a statistically significant relationship

between a higher anti-Toxoplasma IgG endpoint titer

and higher seroprevalence level per age group

(x2 = 21.43; P < 0.0001).

4. Discussion

Worldwide, toxoplasma prevalencies in cats ranges

between 5.4% (Maruyama et al., 2003) and 90%

(Haddadzadeh et al., 2006). In this study we show that in

Belgian house cats up to the age of 7 years, Toxoplasma

seroprevalence increases annually by 6.25%, with an

average seroprevalence of nearly 25%. Few studies have

reported on comparative studies with ELISA for

toxoplasmosis in cats. In general, the Sabin-Feldman

Dye Test (SFDT) and IIFA have a higher sensitivity and

specificity than ELISA or agglutination test (AT) and

have been used as the gold standards to compare other

methods for toxoplasma serodiagnosis of cats. Evalua-

tion of cat sera in ELISA showed a low sensitivity of the

ELISA tests; 84.1% and 88.6% of IIFA positive cats

ELISA

Anti-SAG1b Anti-TLAb IgG endpoint titer against TLAc

n.d. n.d. n.d.

42.9% 66.7% 53.600 � 15.006 (6.400–102.400)

71.4% 85.7% 11.520 � 3.732 (6.400–25.600)

100% 100% 14.933 � 5.088 (3.200–51.200)

75.0% 78.6% 8.533 � 1.903 (3.200–25.600)

92.3% 92.9% 22.171 � 7.504 (3.200–102.400)

90.0% 100% 46.484 � 14.073 (6.400–204.800)

87.5% 87.5% 22.400 � 4.538 (6.400–51.200)

84.1%f 88.6%f 27.536 � 4.250g

ge. The endpoint titer of seropositive reference serum was >409.600.

e samples.

. not determined as only one cat serum was positive.

S. De Craeye et al. / Veterinary Parasitology 157 (2008) 128–132 131

reacted to SAG1 and TLA, respectively. SAG1 is an

abundant immunodominant protein located on the

surface of T. gondii tachyzoites (Kasper et al., 1983).

Previously, recombinant SAG1 was evaluated in an

ELISA for cats and results were in agreement with a

latex AT (Kimbita et al., 2001). Compared to IIFA,

sensitivity and specificity of recombinant antigen based

ELISA were 92.3% and 96.4% (Dabritz et al., 2007a),

and 95% and 99%, respectively (Tenter et al., 1994).

In Belgian stray cats, 70.2% tested seropositive for T.

gondii (Dorny et al., 2002). About 50% of cats, aged 0–2

years, from the city of Antwerp, Belgium, tested

seropositive for T. gondii. 2.7% of these cats, the most

at1yearofagewereactivelysheddingoocysts (VanBeeck

etal., 1985). Inotherstudies,only0.28–0.9%ofhouseand

stray cats were found to shed oocysts (Childs and Seegar,

1986; Dabritz et al., 2007b; Svobodova et al., 1998). Only

one out of 567 tested cats (0.18%) showed IgM without

IgG antibodies, which suggests infection occurred 1–2

weeks before sampling and that the cat might have been

shedding oocysts at the time of sampling. The increase of

IgM prevalence at age 7 may be linked to age related

parameters (Campbell et al., 2004). Many studies have

shown that experimentally infected cats are immune to

oocyst shedding when re-exposed to T. gondii (Chavkin

etal.,1994;DavisandDubey,1995;Dubey,1995;Frenkel

and Smith, 1982; Freyre et al., 2007; Lappin et al., 1996).

However, this immunity is waning as cats were

susceptible for oocyst shedding 6 years after the primary

infection(Dubey,1995).Noevidence isavailablewhether

naturally infected cats will reshed Toxoplasma oocysts

later in life. Oocysts can remain infectious for more than

200 days in moderate temperatures and humidity (Dubey,

1998) and when dispersed in the living-area this might

pose a long-term risk of exposure to his human co-

habitants. Recent data shows that environmental oocyst

contamination is concentrated in and around defecation

sites (Afonso et al., 2008).

In our study, we found a significant correlation

between the presence of high antibody titers and higher

seroprevalencies at ages 1 and 6, certainly reflecting a

primary infection in young cats aged 1 year and

suggesting that re-infection with T. gondii might have

occurred in 6-year old cats. Peak Toxoplasma-specific

IgG antibody titers have been observed shortly after

experimental infection which subsequently declined,

but all cats remained seropositive (Dubey et al., 1995).

Cats may become infected when their owner offers

them raw meat containing tissue cysts as a treat, or when

house cats with outdoor access ingest oocysts from the

environment or by occasionally predating wildlife

(Afonso et al., 2006; Woods, 2003).

In this study we show that Toxoplasma infection in

Belgian house cats is quite common. The seropreva-

lence levels increased gradually with age. Seropreva-

lence peaks were observed in cats aged 1 and 6 years,

and coincided with considerably higher IgG endpoint

titers, indicating a primary infection and suggesting a

possible re-infection with T. gondii at these ages.

Acknowledgements

This work was supported by the Belgian Federal

Public Service for Health, Food Chain Safety and

Environment. We also thank ‘‘Les Amis de l’Institut

Pasteur de Bruxelles’’ a.s.b.l. for their support.

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