Serological survey of Toxoplasma gondii infection in domestic

cats from northeastern Portugal

Ana Patrıcia Lopes a,b, Luıs Cardoso a,b,c,*, Manuela Rodrigues a,b

a Department of Veterinary Sciences, University of Tras-os-Montes e Alto Douro, P.O. Box 1013, 5001-801 Vila Real, Portugalb CECAV – Veterinary and Animal Science Research Centre, University of Tras-os-Montes e Alto Douro,

P.O. Box 1013, 5001-801 Vila Real, Portugalc Parasite Disease Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto,

Rua do Campo Alegre 823, 4150-180 Porto, Portugal

Received 18 December 2007; received in revised form 18 April 2008; accepted 6 May 2008

www.elsevier.com/locate/vetpar

Available online at www.sciencedirect.com

Veterinary Parasitology 155 (2008) 184–189

Abstract

Cats are very important hosts in the epidemiological cycle of Toxoplasma gondii, a zoonotic protozoan parasite that can infect

humans and many other animal species worldwide. We report a serological survey of antibodies to T. gondii in domestic cats from

northeastern Portugal, by means of the modified agglutination test. Three cats had titres of 20 (3.9%), 18 had titres of 40 (23.7%) and

55 animals had titres of �800 (72.4%). Results of three seropositive kittens with less than 4 months were not considered for

determining the seroprevalence of infection, which was found to be 35.8% (73/204). Differences in the seroprevalence levels were

not statistically significant between males (35.6%) and females (36.0%) or pure non-European (26.7%) and European or mixed-

breed cats (39.6%). Animals aged 36–71 months and 72–180 months had the highest seroprevalences of infection, i.e. 51.7% and

51.2%, respectively, which significantly differ from the values observed in cats with 2–11 months (14.6%) and 12–35 months

(26.3%). Infection levels were also significantly different between cats that lived totally indoors (7.7%) and those that had access to

outdoors (45.4%), as well as between cats living alone (13.8%) and those that had contact with other cats (39.4%). Seroprevalence

values in cats fed only commercial canned or dried food (22.9%) and animals whose diet included raw or undercooked viscera and/

or meat (53.5%) were also significantly different. Furthermore, considering only 108 cats, differences of seropositivity to T. gondii

were significant between feline immunodeficiency virus infected and non-infected animals, but this was not observed for feline

leukaemia virus. Age, habitat and diet were identified as risk factors for the feline T. gondii infection by logistic regression analysis.

Some control measures are suggested based on these findings.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Toxoplasma gondii; Cat; Modified agglutination test; Seroprevalence; Infection; Portugal

* Corresponding author at: Department of Veterinary Sciences,

University of Tras-os-Montes e Alto Douro, P.O. Box 1013,

5001-801 Vila Real, Portugal. Tel.: +351 259 350 458;

fax: +351 259 350 629.

E-mail address: lcardoso@utad.pt (L. Cardoso).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.05.007

1. Introduction

Toxoplasmosis is an endemic parasitic zoonosis

throughout the world. Its etiological agent, the

protozoan Toxoplasma gondii, can infect almost all

the homeothermic animals, including human beings

(Ajzenberg et al., 2004; Dubey, 2004). Infection in

immunocompetent people is mainly subclinical; but in

congenitally infected children and in immunocompro-

A.P. Lopes et al. / Veterinary Parasitology 155 (2008) 184–189 185

mised persons, such as HIV-infected individuals,

toxoplasmosis may be the cause of high morbidity

and mortality rates (Belanger et al., 1999; Avelino et al.,

2003). Furthermore, the disease is also of economic

importance in animal production, because it might

produce abortions and neonatal death, especially in

sheep (Buxton, 1990; Tenter et al., 2000).

Consumption of undercooked meat from mammals or

birds harbouring T. gondii cysts was identified as the

major risk factor for infection in humans (Kapperud et al.,

1996). However, besides being the only way of

transmission to herbivorous animals, ingestion of

sporulated oocysts also contributes in a significant

manner to human infection (Dubey, 2004). As the only

recognised definitive hosts of the parasite, cats and some

wild felids play an important role in the spread of T.

gondii infection, excreting millions of resistant oocysts in

their faeces into the environment (Davidson, 2000). In

spite of that, oocysts are rarely found in the faeces of cats,

because their elimination does not last for more than 1–3

weeks in the animal’s lifetime (Tenter et al., 2000).

Since seropositive cats are likely to have already

shed T. gondii oocysts, serologic surveys for the

detection of anti-T. gondii antibodies in these animals

are helpful to assess the degree of environmental

contamination, as well as to determine the potential risk

of infection in distinct geographical areas (Lucas et al.,

1999; Miro et al., 2004). The modified agglutination test

(MAT) has proved to be the most sensitive and specific

assay for the serological diagnosis of feline toxoplas-

mosis (Dubey and Thulliez, 1989; Patton et al., 1991).

In recent years, many serologic surveys of T. gondii

infection in cats have been reported from Europe

(Dorny et al., 2002; Gauss et al., 2003; Miro et al., 2004;

Meunier et al., 2006), but relatively less is known about

the feline infection in Portugal. The present work aimed

at determining the seroprevalence of T. gondii infection

in domestic cats from the region of Tras-os-Montes e

Alto Douro (northeastern Portugal), using the MAT, as

no studies on feline toxoplasmosis have been reported

from this part of the country.

2. Materials and methods

2.1. Animals and samples

Serum samples were obtained from 207 domestic

cats of the region of Tras-os-Montes e Alto Douro, in

northeastern Portugal, between May 2004 and May

2005. One hundred and sixty-one animals were from the

municipality of Vila Real (418170N, 78480W) and the

remaining 46 cats were from 10 other contiguous

municipalities. Questionnaires were provided to owners

requesting data about each sampled animal. Collected

data included information on gender, breed (pure non-

European breed or European breed and mixed-breed),

age, habitat (whether the cats lived totally indoors or

had access to outdoors), whether they had contact with

other cat(s), and the type of diet received (whether the

animals were fed strictly on commercial canned or dried

food, or they also ate other types of food including raw

or undercooked viscera and/or meat). Data on the

serological assessment of feline immunodeficiency

virus (FIV) and feline leukaemia virus (FeLV) were

obtained for 108 cats.

2.2. Modified agglutination test

The serum samples were analysed for antibodies to

T. gondii with the MAT using a direct microagglutina-

tion commercial kit (Toxo-Screen DA1, bioMerieux,

Lyon, France). Sera were tested at the dilutions of 1:20,

1:40 and 1:800, with whole tachyzoites as antigen and

the addition of 2-b-mercaptoethanol (Desmonts and

Remington, 1980). Positive and negative control

samples were included in each plate. Results obtained

with the MAT were expressed as an antibody titre, i.e.

the reciprocal of the highest dilution at which

agglutination (at least one half of the well’s diameter)

was still visible after 5–18 h incubation at room

temperature. A cut-off titre of 20 was chosen to

maximize both sensitivity and specificity of the test

(Dubey et al., 1995b).

2.3. Data analysis

Chi-square or Fisher’s exact tests were used to

compare seroprevalence values relative to gender, age,

breed, habitat, contact with other cats, diet, FIV and

FeLV. Multiple logistic regression was used to identify

independent risk factors for the prevalence of anti-T.

gondii antibodies, calculating odds ratios (OR) and their

95% confidence intervals (CI). Analyses were done with

SPSS 10.0 software for Windows, with a probability ( p)

value <0.05 as statistically significant. Confidence

limits for the proportions were established by exact

binomial test with 95% CI (Altman, 1991).

3. Results

Seventy-six out of the 207 domestic cats studied

were seropositive using a cut-off value of 20, which

proved that they had been exposed to T. gondii infection.

Three seropositive cats had titres of 20 (3.9%); 18 had

A.P. Lopes et al. / Veterinary Parasitology 155 (2008) 184–189186

Table 1

Seroprevalence of Toxoplasma gondii infection in cats (n = 204) from northeastern Portugal, by gender, breed, age group, habitat, possible contact

with other cat(s), and type of diet received

Animals tested (n) Relative distribution (%) MAT-positive (n) Prevalence (%) 95% CI

Gender

Male 104 51.0 37 35.6 26.4–45.6

Female 100 49.0 36 36.0 26.6–46.2

p = 1.000

Breed

Pure non-European breed 60 29.4 16 26.7 16.1–39.7

European or mixed-breeds 144 70.6 57 39.6 31.5–48.1

p = 0.111

Age (months)

[2–11] 48 23.5 7 14.6 6.1–27.8

[12–35] 57 27.9 15 26.3 15.5–39.7

[36–71] 58 28.4 30 51.7 38.2–65.0

[72–180] 41 20.1 21 51.2 35.1–67.1

p < 0.001

Habitat

Totally indoors 52 25.5 4 7.7 2.1–18.5

Access to outdoors 152 74.5 69 45.4 37.3–53.7

p < 0.001

Contact with other cat(s)

No 29 14.2 4 13.8 3.9–31.7

Yes 175 85.8 69 39.4 32.1–47.1

p = 0.014

Diet

Strictly commercial food 118 57.8 27 22.9 15.6–31.5

Including raw or undercooked

viscera and/or meat

86 42.2 46 53.5 42.4–64.3

p < 0.001

Total 204 100 73 35.8 28.6–42.0

titres of 40 (23.7%); and 55 animals had titres of �800

(72.4%). Seven kittens aged less than 4 months were

seropositive: four had titers�800 and three had titers of

40. The results of these three kittens were not

considered for determining the seroprevalence of

infection and for the subsequent statistical analysis.

Table 2

Seroprevalence of Toxoplasma gondii infection in cats (n = 108) from northe

feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV)

Animals tested (n) Relative distribution (%)

FIV

Negative 92 85.2

Positive 16 14.8

FeLV

Negative 101 93.5

Positive 7 6.5

Total 108 100

The studied animals were aged between 2 months

and 15 years (180 months), with a median value of 31.5

months (interquartile range: 12.0–60.0). For statistical

purposes, four groups were designed based on an equal

distribution of cats according to age. Results regarding

the distribution of sampled animals (n = 204) and the

astern Portugal, according to the results of serological assessment for

MAT-positive (n) Prevalence (%) 95% CI

33 35.9 26.1–46.5

12 75.0 47.6–92.7

p = 0.008

40 39.6 30.0–49.8

5 71.4 29.0–96.3

p = 0.125

45 41.7 32.2–51.5

A.P. Lopes et al. / Veterinary Parasitology 155 (2008) 184–189 187

Table 3

Multiple logistic regression to identify risk factors for the seroprevalence of Toxoplasma gondii infection in cats (n = 204) from northeastern

Portugal

Animals tested (n) Prevalence (%) OR 95% CI

Age (months)

[2–11] 48 14.6 1.0

[12–35] 57 26.3 2.55 0.87–7.47

[36–71] 58 51.7 8.74 3.03–25.22

[72–180] 41 51.2 7.89 2.57–24.29

p < 0.001

Habitat

Totally indoors 52 7.7 1.0

Access to outdoors 152 45.4 6.83 2.12–21.98

p = 0.001

Contact with other cat(s)

No 29 13.8 1.0

Yes 175 39.4 1.41 0.38–5.22

p = 0.611

Diet

Strictly commercial food 118 22.9 1.0

Including raw or undercooked viscera and/or meat 86 53.5 3.19 1.57–6.47

p = 0.001

respective seroprevalence values by gender, breed, age,

habitat, contact with other cat(s) and diet are presented

in Table 1. The overall seroprevalence was 35.8% (95%

CI: 28.6–42.0).

Table 2 presents values of anti-T. gondii antibodies

prevalence in those cats (n = 108) assessed for FIV and

FeLV infections.

For multiple logistic regression only those indepen-

dent variables that presented p < 0.05 in the chi-square

analysis were considered (Table 3), with the exception

of FIV infection due to the lower number of animals

sampled. Risk factors for T. gondii infection in cats were

age, habitat and diet; contact with other cat(s) was

shown not to be a statistically significant risk factor in

logistic regression analysis.

4. Discussion

Information on the prevalence of infection in cats is

useful for assessing environmental contamination by

the protozoan and the associated risk for public health

(Lucas et al., 1999; Dubey, 2004).

The present work revealed antibodies to T. gondii in 76

out of 207 domestic cats, which prove these animals’

exposure to T. gondii infection. Four out of the seven

seropositive kittens aged less than 4 months – the age at

which collostraly acquired anti-T. gondii antibodies

disappear (Dubey et al., 1995a) – had titres of�800, and

three other had titres of 40. Due to the high levels of

serum antibodies, which do not match with a hypothetical

colostral transmission and further disappearance in a

short-term, kittens with a titre of �800 were most

probably infected with T. gondii (Miro et al., 2004).

Regarding the three kittens aged less than 4 months and

with a titre of 40, it was not possible to be sure about the

source of these antibodies, i.e. whether they had been

received colostrally or resulted from infection acquired

congenitally or after birth (Dubey, 1973). This was the

reason why these three kittens were excluded from the

determination of seroprevalence by group and from the

assessment of risk factors for infection.

This serological survey on T. gondii infection in cats

represents a considerable geographical area of 11

municipalities from the northeastern part of Portugal.

Taking into account the premise stated above for the

three kittens, in the present study seroprevalence of

infection was 35.8% (73/204). The seropositive cats

observed in the present study are likely to have already

shed oocysts into the environment (Tenter et al., 2000).

The present work did not find a significant difference

between seroprevalences in male and female cats,

which is in line with data obtained from several other

studies in different European countries (D’Amore et al.,

1997; Dorny et al., 2002; Smielewska-Los and Pacon,

2002). These observations suggest that both males and

females are equally exposed and susceptible to

infection. Additionally, cat’s breed seemed not to

influence the prevalence of T. gondii infection. In our

study, European and mixed-breed cats, the most

common breeds found in northeastern Portugal, had a

A.P. Lopes et al. / Veterinary Parasitology 155 (2008) 184–189188

higher but non-significantly different seroprevalence

than pure non-European breeds. These results are

quantitatively compatible with those from Gauss et al.

(2003), who found higher seroprevalences in European

and mixed-breeds than in pure breed non-European

cats. On the contrary, Svoboda and Svobodova (1987)

reported that the seropositivity to T. gondii in Siamese

and Persian cats was higher than that found in European

short-haired cats, with the difference being significant.

According to these authors, a hypothetical explanation

for the lower seropositivity in European cats could be

their better adaptability to the local environmental

conditions.

In the present study, risk factors for T. gondii infection

in cats were shown to be age, habitat and diet (Table 3).

Most studies from several other authors have widely

assessed differences on seroprevalence values between

groups of cats but did not quantify risk factors for

infection (Svoboda and Svobodova, 1987; Uggla et al.,

1990; D’Amore et al., 1997; Miro et al., 2004; Meunier

et al., 2006). In our study, a significant difference was

observed between cats that had contact with other cat(s)

and those living alone. However, contact with other cat(s)

was not identified as a valid risk factor for infection after

multiple logistic regression analysis. In Barcelona,

Spain, a significant difference between the seropositivity

values of cats living in a community of more than five cats

(feral or domestic) and cats living alone (domestic) was

reported by Gauss et al. (2003), but no quantification of

risk factors was done.

The increased infection levels found in the two

groups of older cats from our study, i.e. those animals

aged 36 months or above, are probably related to a more

prolonged exposure of adult cats to the T. gondii

infective forms in the course of their lives. Although

based on variable age groups that do not absolutely

coincide with ours, the same type of observations

regarding higher seropositivity in adult (�12 months) or

older cats have been reported by several other authors

(Tenter et al., 1994; Gauss et al., 2003; Miro et al., 2004;

Afonso et al., 2006).

In the present study, the higher seroprevalence in cats

with access to outdoors can be explained by an increased

probability of these animals ingesting oocysts from the

environment or mainly tissue cysts in intermediate hosts.

In fact, cats living outdoors have the opportunity to hunt

and eat small mammals or birds, especially when acting

in groups (Gauss et al., 2003). A significant difference in

the seropositivity of animals with or without access to the

outdoors was also observed by Tenter et al. (1994).

Additionally, feral and stray cats had a significantly

higher prevalence of antibodies to T. gondii when

compared to household animals, as reported by Gauss

et al. (2003) and Miro et al. (2004).

Higher seroprevalences have been referred in cats

that are fed on diets including raw or undercooked

viscera and/or meat in comparison with animals

receiving only commercial canned or dried food

(Svoboda and Svobodova, 1987). In our study, a higher

percentage of infection was also recorded in the former

type of cats. This higher frequency of infection can most

likely be related to the ingestion of viable T. gondii cysts

in uncooked or undercooked infected meat. Diet seems

to greatly influence infection levels, and even cats that

are kept strictly indoors can have a marked contact with

the parasite due to their feeding habits (Gauss et al.,

2003). Heating meat to an internal temperature of 67 8Cor freezing it to less than�12 8C kills tissue cysts of the

parasite (Kotula et al., 1991; Dubey, 1996).

FIVand FeLV infections were assessed because these

viruses are immunosuppressive (D’Amore et al., 1997).

One possible reason why FIV positive cats had a

significantly different seropositivity to T. gondii is that

the virus leads to a transient proliferation of encysted

bradyzoites resulting in increased antigenemia and

stimulation of specific humoral immunity (Lappin et al.,

1992).

In conclusion, T. gondii infection in domestic cats is

highly prevalent in northeastern Portugal. Taking into

account the quantitative risk factors identified in the

present study, control measures must include not

allowing animals to live or stay outdoors, to prevent

them from hunting. Feeding cats with commercial diets

or with food processed either by cooking or freezing

must also be put into practice. With the ultimate goal of

preventing infection in human beings, cats and other

domestic animals, control of the zoonotic infection

should be based on an integrated sanitary approach over

definitive but also intermediate hosts.

Acknowledgements

The authors would like to thank their colleagues of

the veterinary medical staff at the Veterinary Teaching

Hospital, University of Tras-os-Montes e Alto Douro,

and cat owners for their willingness to cooperate with

this study. Thanks are extended to Cristina Abraao and

Teresa Coutinho for assistance in sample collection.

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