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Veterinary Parasitology 174 (2010) 37–42

Contents lists available at ScienceDirect

Veterinary Parasitology

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ow seroprevalence of Leishmania infantum infection in cats fromorthern Portugal based on DAT and ELISA

uís Cardosoa,b,1, Ana Patrícia Lopesa,1, Kate Sherryc, Henk Schalligd,aia Solano-Gallegoc,∗

Department of Veterinary Sciences, University of Trás-os-Montes e Alto Douro, P.O. Box 1013, 5001-801 Vila Real, PortugalParasite Disease Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, PortugalDepartment of Pathology and Infectious Diseases, Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA,nited KingdomKIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute), KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands

r t i c l e i n f o

rticle history:eceived 15 June 2010eceived in revised form 30 July 2010ccepted 16 August 2010

eywords:atirect agglutination testLISAeishmania infantumortugaleroprevalence

a b s t r a c t

Cats have been considered playing a role in the epidemiology of leishmaniosis caused byLeishmania infantum, an endemic zoonosis in countries of the Mediterranean basin. Thepresent study assessed the prevalence of antibodies to L. infantum in 316 domestic catsfrom northern Portugal, by means of the direct agglutination test (DAT) and an enzyme-linked immunosorbent assay (ELISA). Seroreactivity to DAT was found in six cats, andnine cats were positive in the ELISA, including the six DAT-positive animals. The overallseroprevalence of Leishmania infection was 2.8%, based on ELISA and DAT. A substantialagreement (99%; � value = 0.80) was found between DAT and ELISA results. The differencebetween seroprevalence values in females (0.7%) and males (4.7%) was statistically sig-nificant (p = 0.045). The age of seropositive cats ranged from 31 to 84 months. Cats with5–6 years (60–71 months) and 6–7 years (72–83 months) presented the highest level ofseropositivity (15.4% and 33.3%, respectively). A significant difference was found comparingthe seroprevalences in cats aged less than 24 months (0.0%) and in those with 24 monthsor more (7.3%) (p = 0.022). Seroprevalences in cats living in a rural environment (10.5%) orin urban areas (0.0%) were also found to be significantly different (p < 0.001). No significantdifferences were detected between serological status to Leishmania in pure non-Europeanand European or mixed breeds (p = 0.442), cats that lived totally indoors and those thathad access to outdoors (p = 0.412), cats seropositive and seronegative to Toxoplasma gondii

(p = 0.276), or apparently healthy animals and those with clinical illness (p = 0.271). Thisstudy is the largest epidemiological investigation performed on feline Leishmania infectionin Portugal. The seroprevalence of Leishmania infection was low in cats living in northernPortugal, a region where canine leishmaniosis is endemic. Nevertheless, Leishmania infec-tion must not be underestimated and leishmaniosis may be included in the differential

ous or

diagnosis of cutane

∗ Corresponding author. Tel.: +44 1707666326; fax: +44 1707661464.E-mail address: lsolano@rvc.ac.uk (L. Solano-Gallego).

1 These two authors contributed equally to this work.

304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2010.08.022

systemic clinical signs in cats.© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Leishmaniosis caused by the protozoan Leishmaniainfantum (syn. Leishmania chagasi) is an endemic zoonoticdisease in the Mediterranean basin and Portugal (WHO,2000). Dogs are the main reservoir of the parasite, whichhas phlebotomine sand fly insects as vectors to other ver-

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38 L. Cardoso et al. / Veterina

tebrate hosts (Baneth et al., 2008). In humans, visceralleishmaniosis is the most severe clinical manifestation ofL. infantum infection, being observed mainly in childrenand immunocompromised adults (Desjeux, 2001). In addi-tion, canine leishmaniosis is a systemic, chronic, and evenfatal clinical condition that represents an important veteri-nary medical and public health problem in southern Europe(Miró et al., 2008). A large majority of the infected dogsdoes not develop clinical signs but they may still be capa-ble of transmitting the parasite to the vectors (Molina et al.,1994).

Sporadic clinical cases of leishmanial infection, withconfirmed or presumed L. infantum parasites, have beenreported in cats from European countries where canineleishmaniosis is endemic, such as Italy (Pennisi, 2002; Poliet al., 2002), France (Ozon et al., 1998), Spain (Hervás etal., 1999) and Portugal (Durão et al., 1994). The uncom-mon clinical cases of feline leishmaniosis are characterizedpredominantly by cutaneous lesions, although visceralinvolvement has also been described in association or assingle manifestations (Pennisi, 2002; Simões-Mattos et al.,2004).

Cats have been suggested as a secondary reservoir inareas where L. infantum is endemic, and it is necessary toevaluate their infection status and role in the epidemiologyof zoonotic leishmaniosis (Mancianti, 2004; Maroli et al.,2007).

Detection of antibodies to Leishmania remains an impor-tant diagnostic tool, and the enzyme-linked immunosor-bent assay (ELISA) and the direct agglutination test(DAT) have proved to be suitable for the evaluation oflarge numbers of serum samples in dogs (Schallig et al.,2002; Santarém et al., 2010). In countries of southernEurope, serological investigations performed among felinepopulations by different techniques have revealed sero-prevalences ranging from less than 1% to more than 60% indifferent regions and groups of animals (Pennisi, 2002; Poliet al., 2002; Martín-Sánchez et al., 2007; Solano-Gallego etal., 2007; Duarte et al., 2010).

In Portugal, the northern Alto Trás-os-Montesand Douro contiguous subregions have the highestendemicity levels for canine leishmaniosis. Local sero-prevalences of Leishmania infection in dogs have beendemonstrated to range between 6.5% and approximately20% (Santos, 1997; Cardoso et al., 2004b). The aim of thepresent study was to assess the prevalence of antibodies toLeishmania in a large and representative sample of domes-tic cats from northern Portugal (Alto Trás-os-Montes andDouro), since no information is available on leishmanialinfection among the feline population.

2. Materials and methods

2.1. Cats and samples

Three-hundred and sixteen domestic cats from

northern Portugal (Alto Trás-os-Montes and Dourosubregions) were investigated on the basis of serumsamples obtained year-round between May 2004 and July2008. Two-hundred and forty two cats were assayed at theVeterinary Teaching Hospital of the University of Trás-os-

itology 174 (2010) 37–42

Montes e Alto Douro (VTH-UTAD) and the remaining 74 attheir owner’s home.

Additional available data included gender (n = 306),breed (n = 190), age (n = 189), urban or rural origin (n = 193),indoors or mixed habitat (n = 173) and health status basedon physical examination and clinical history (n = 165).Cats were categorized as young (2–11 months), adult(12–83 months) or old (84–204 months); and of purenon-European or European and mixed breeds.

Serological assessment of antibodies to Toxoplasmagondii, with Toxo-Screen DA® (bioMérieux, Lyon, France),was also available for 280 cats, at a cut-off titre of 20 (Lopeset al., 2008).

2.2. Direct agglutination test – DAT

The DAT for titration of antibodies specific to Leishmaniafollowed the general procedures described by Schalliget al. (2002), using a standard freeze-dried antigen at aconcentration of 5 × 107 promastigotes/ml (KIT BiomedicalResearch, Amsterdam, The Netherlands). Briefly, the serumsamples were diluted in 0.9% NaCl saline containing 1.56%�-mercaptoethanol. Twofold dilution series were maderanging from 1:100 to 1:102,400 in V-shaped microtitreplates (Greiner, Germany) and incubated for 1 h at 37 ◦C.Fifty microlitres of reconstituted DAT antigen was sub-sequently added to each well containing 50 �l of dilutedserum. Results obtained with DAT are expressed as anantibody titre, i.e. the reciprocal of the highest dilutionat which agglutination (large diffuse blue mats) is stillclearly visible after 18 h incubation at room temperature.To maximize sensitivity of the test, cats were consideredseropositive at a dilution of 1:100.

Positive controls for the DAT consisted of serumsamples from four cats with leishmaniosis (provided byProfessor Maria Grazia Pennisi, Department of Veteri-nary Medical Science, Faculty of Veterinary Medicine,University of Messina, Italy). Individual DAT titres rangedbetween 3200 and 25,600 in these samples. Sera fromfour other cats living in areas where leishmaniosis is notendemic were used as negative controls.

2.3. Enzyme-linked immunosorbent assay – ELISA

An ELISA protocol previously described for cat sera(Solano-Gallego et al., 2007) was used with some modifi-cations (Sherry et al., 2010). Briefly, microtitre plates werecoated with 100 �l of L. infantum (MHOM/FR/78/LEM-75zymodeme MON-1) antigen (1 mg in 50 ml of 0.05 Mcarbonate–bicarbonate, pH 9.6) and incubated overnightat 4 ◦C. One hundred microlitres per well of cat sera, diluted1:100 in PBS–0.05% Tween 20 (PBST)–1% dried skimmedmilk (PBST-M), was incubated for 1 h at 37 ◦C. After threewashes with PBST and one wash with PBS, 100 �l perwell of anti-cat IgG (AbD, Serotec, Kidlington, UK) diluted1:7500 in PBST-M conjugated to horseradish peroxidase

was added and incubated for 1 h at 37 ◦C. Then, the plateswere rewashed as above. The substrate solution of ortho-phenylenediamine dichloride tablets, 0.4 mg/ml (SIGMAFASTTM, Sigma–Aldrich, St. Louis, USA), plus 0.4 �l/mlurea hydrogen peroxide in 0.05 M phosphate–citrate,

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H 5.0, was added at 200 �L per well and developed for0 min at 24 ◦C. The reaction was stopped with 50 �l ofM H2SO4. Absorbance values were read at 490 nm inn automatic micro-ELISA reader (Spectra Max M2 Plateeader, Molecular Devices, Wokingham, UK). The reactionas quantified as ELISA units (EU) related to a positive

at sera used as a calibrator and arbitrarily set at 100U. The calibrator cat had confirmed leishmaniosis anderum from this cat has been used in previous studiesSolano-Gallego et al., 2007). All determinations includedhe calibrator serum as a positive control and serum ofcat from the UK as a negative control. The cut-off was

stablished at 69 EU for IgG (mean + 4 standard deviationsf sera of 76 cats from the UK cat population).

.4. Data analysis

The agreement beyond chance between DAT and ELISAesults was assessed with Cohen’s kappa value (�). Sero-revalence values to L. infantum relative to gender, breed,ge, origin, habitat, serology to T. gondii and clinical sta-us were compared by means of Fisher’s exact test, andonfidence limits for the proportions established by exactinomial test with 95% confidence intervals (CI). Analysesere done with SPSS 11.5 software for Windows, with arobability (p) value <0.05 as statistically significant (Petriend Watson, 1999).

. Results

Data on the characterization of cats and seroprevalencealues are presented in Table 1. The cats were aged betweenmonths and 17 years (204 months), with a median of

2 months (interquartile range: 12–60). Seroreactivity toAT antigen was found in six cats, at the dilutions of 1:100

n = 4) and 1:200 (n = 2). Nine cats were found positive inhe ELISA (70–109 EU), including those six DAT-positivenimals. The observed proportion of agreement betweenesults of the two serological tests was 0.99, with a � valuef 0.80, which represents a substantial agreement beyondhange. Considering that nine cats were seropositive, eithery DAT or ELISA, the overall seroprevalence of Leishmania

nfection was set at 2.8%.A statistically significant difference was found between

he seroprevalence values in females and males (p = 0.045).he age of seropositive cats ranged from 31 to 84 months.ats with 5–6 years (60–71 months) and 6–7 years (72–83onths) presented the highest level of seropositivity

15.4% and 33.3%, respectively). No significant differencesere detected when comparing young (2–11 months) and

dult (12–83 months) (p = 0.191), young and old (84–204onths) (p = 0.389), or adult and old animals (p = 1.0).owever, a significant difference (p = 0.022) was foundomparing the seroprevalences in cats aged less than4 months (0.0%) and in those with 24 months or more7.3%). The difference between the seroprevalences in cats

iving in a rural environment (villages or towns) and thoseiving in urban areas (cities) was found to be statisticallyignificant (p < 0.001). On the other side, no significantifferences were detected between serological status toeishmania in pure non-European and European or mixed

itology 174 (2010) 37–42 39

breeds (p = 0.442), cats that lived totally indoors and thosethat had access to outdoors (p = 0.412), cats seropositiveand seronegative to T. gondii (p = 0.276), or apparentlyhealthy animals and those with clinical illness (p = 0.271).

4. Discussion

The present study is the first epidemiologicalinvestigation performed on feline Leishmania infec-tion in northern Portugal and the largest one carried out inthe whole country. A low seroprevalence of infection withLeishmania was revealed, with higher levels in males, oldercats and those living in a rural environment. Furthermore,a substantial agreement was found between DAT and ELISAresults, with the use of these two tests being reported forthe first time in combination to diagnose feline Leishmaniainfection.

A wide range of specific seroprevalences has beenreported in cats from countries in the Mediterraneanbasin where canine leishmaniosis is endemic. Regardingthe Iberian Peninsula, the 2.8% seroprevalence from thepresent study is lower than the 5.3–6.3% values found bySolano-Gallego et al. (2007) in 445 cats from northeasternSpain, the 20% seropositivity found by Maia et al. (2008) in20 female stray cats from Lisbon (southern Portugal), the28.3–60% found by Martín-Sánchez et al. (2007) in 183 catsfrom southern Spain, and the 42% found by Zárate-Ramoset al. (2002) in 50 cats from the northeastern part of Spain;is line with the values of 1.3–4.3% found by Ayllon etal. (2008) in 233 cats from central Spain; but is higherthan the 0.6% found by Duarte et al. (2010) in 180 strayfrom the metropolitan area of Lisbon. On the other side,in Italy, Poli et al. (2002) found a 0.9% seroprevalence in110 cats, Vita et al. (2005) a 16.3% value in 203 cats, andPennisi (2002) 61% in 93 and 68% in 89 cats. In Greece,Diakou et al. (2009) found a 3.9% seroprevalence in 284cats.

The different results from the same or distinct geo-graphical regions may reflect the real endemicity levelsof Leishmania infection among the local feline populations,but might also express the influence of other determinants.The latter comprise differences in the sampled populations,including the health status of cats (Pennisi, 2002), as well asdifferences in the serological techniques and their cut-offvalues or positivity thresholds (Zárate-Ramos et al., 2002).Moreover, based on previous studies, the seroprevalence ofLeishmania infection in cats has been found lower than indogs from the same areas (Poli et al., 2002; Solano-Gallegoet al., 2007; Diakou et al., 2009). This is also the case ofnorthern Portugal where the prevalence of antibodies toLeishmania in dogs is up to 20% or more (Cardoso et al.,2004a,b) and significantly different from the one deter-mined here in cats (2.8%).

In the present study, a significant difference betweenjuvenile or young adults (2–23 months) and adult orold cats (24–204 months) regarding seroprevalence may

probably be explained by a cumulative exposure of olderanimals to infection. On the other side, the differencebetween female and male cats is difficult to explain(Table 1). Other authors have not found such differencesof seroprevalence regarding gender (Solano-Gallego et al.,

40 L. Cardoso et al. / Veterinary Parasitology 174 (2010) 37–42

Table 1Seroprevalence of L. infantum infection in cats from northeastern Portugal by gender, breed, age group, origin, habitat, serology to T. gondii and clinicalstatus.

Cats tested(n)

Relativedistribution (%)

DAT and/or ELISApositive (n)

Seroprevalence ofLeishmania infection (%)

95% CI

Gender 306 p = 0.045Female 137 44.8 1 0.7 0.0–4.0Male 169 55.2 8 4.7 2.1–9.1

Breed 190 p = 0.422Pure non-European 55 28.9 1 1.8 0.0–9.7European or mixed 135 71.1 7 5.2 2.1–10.4

Age group 189 p > 0.05a

Young 44 23.3 0 0.0 0.0–8.0Adult 117 61.9 7 6.0 2.4–11.9Old 28 14.8 1 3.6 0.1–18.3

Origin 193 p < 0.001Urban 117 60.6 0 0.0 0.0–3.1Rural 76 39.4 8 10.5 4.7–19.7

Habitat 173 p = 0.412Totally indoors 43 24.9 3 7.0 1.5–19.1Access to outdoors 130 75.1 5 3.8 1.3–8.7

T. gondii 280 p = 0.276Negative 201 71.8 5 2.5 0.8–5.7Positive 79 28.2 4 5.1 1.4–12.5

Clinical status 165 p = 0.271Apparently healthy 110 66.7 7 6.4 2.6–12.7

1

9

Sick 55 33.3

Total 316 100

a Comparison performed with pairs of groups.

2007; Ayllon et al., 2008; Diakou et al., 2009). Cats comingfrom a rural environment had a higher seroprevalence ofinfection, with the difference being significant in compari-son with those animals from urban areas. This may be dueto a higher rate of exposure to vectors of Leishmania in therural areas (villages or towns). In the present study, thehypotheses that males were more exposed by being olderor living outdoors were not statistically supported. How-ever, the fact that 47.0% of the males and only 31.5% offemales were originated from a rural environment, with thedifference being statistically significant (data not shown),may contribute to explain the overall differences in theseroprevalence according to gender.

ELISA (Solano-Gallego et al., 2007; Nasereddin et al.,2008; Diakou et al., 2009) and immunofluorescence assays(IFA) (Poli et al., 2002; Martín-Sánchez et al., 2007; Ayllonet al., 2008) have been used for the detection of antibodiesto Leishmania in cats. The DAT is often used to detectantibodies to Leishmania in humans and dogs (Schallig etal., 2002), but there are fewer reported attempts of its usein cats (Zárate-Ramos et al., 2002; Sarkari et al., 2009). Inthe present study, the titres or concentrations of antibodiesto Leishmania detected in cats, by either DAT or ELISA,were much lower than those commonly observed in dogs.This circumstance demanded a fourfold and a 2.7-foldlower dilutions of serum and conjugate, respectively, inthe ELISA (Solano-Gallego et al., 2001) and a fourfold lowercut-off titre in the DAT (Schallig et al., 2002), in comparisonwith the same serological tests performed for dogs. With

the exception of the positive controls, none of the nineseropositive cats reached the usual DAT cut-off titre of400 for canine Leishmania infection. Specificity of theserological assessment was strengthened by the fact thatno significant differences were detected between the sero-

1.8 0.0–9.7

2.8 1.3–5.3

prevalences of Leishmania infection in cats seropositive orseronegative to T. gondii (Nasereddin et al., 2008; Sherryet al., 2010). Furthermore, the seroprevalence of T. gondiiinfection in the present study is consistent with a previoussurvey in the same area (Lopes et al., 2008). DAT and ELISAresults from the present study support the assumptionthat the immune response to Leishmania infection in cats isdifferent from the one observed in dogs. A higher degree ofnatural resistance to infections with the protozoan in catsis probably determined by the feline genetic backgroundand related immune responses (Mancianti, 2004).

The polymerase chain reaction (PCR) has also beenused, alone or in combination with serology, to assessthe prevalence of feline Leishmania infection. In general,seroprevalence is higher than PCR-positivity to Leishmaniain cats, although the detection of parasitic DNA in seroneg-ative animals may give rise to an overall increased levelof positivity. Martín-Sánchez et al. (2007) found 25.7%PCR-positivity and a 70.6% total positivity (maximumseropositivity of 60%); and Ayllon et al. (2008) 0.43%PCR-positivity for a 1.7–4.7% total positivity (1.3–4.3%seropositivity). Also in Spain (Ibiza island), Sherry et al.(2010) found 8.7% PCR-positivity and a 15.4% total positiv-ity (maximum seropositivity of 13.2%). On the other side,Maia et al. (2008) found 30.4% PCR-positivity and 34.8% oftotal positivity (seropositivity of 20%). Additionally, Tabaret al. (2008), in 100 cats from northeastern Spain, founda prevalence of 3% by PCR alone, and proposed that sero-prevalence could overestimate the real or active degree of

infection. In the present study, infection in cats was testedonly by means of serology and an underestimation of theexposure to Leishmania may be possible.

Infection and disease caused by Leishmania in domes-tic cats seem to be rare in northern Portugal. To our

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nowledge, only three sporadic clinical cases of felineeishmaniosis have been reported in Portugal (Durão et al.,994; Marcos et al., 2009; Sanches et al., 2009), but none

n the region where the present study was conducted.his finding may confirm a low susceptibility of cats toeishmania infection. The leishmanial species infectingats in northern Portugal is most probably L. infantum (syn.. chagasi). In the Douro subregion, L. infantum zymodemesON-1 has been demonstrated in humans, dogs and

hlebotomus ariasi, MON-24 in P. ariasi and MON-98 inogs (Cardoso et al., 2002; Campino et al., 2006).

Information on the prevalence of L. infantum infections necessary to define control measures for zoonotic leish-

aniosis. Although dogs are the main reservoir of thearasite in countries of the Mediterranean basin includingortugal, the role of cats in the epidemiology of L. infantumeeds further attention.

In conclusion, the seroprevalence of Leishmania infec-ion is low in cats living in northern Portugal, a regionhere zoonotic leishmaniosis is endemic. Nevertheless,

eishmania infection must not be underestimated andeishmaniosis may be included in the differential diagnosisf cutaneous or systemic clinical signs in cats.

cknowledgements

The authors thank Prof. M.G. Pennisi and Dr. A.odríguez Cortés, for kindly supplying positive serum con-rols; Dr. F.T. Rodrigues, Dr. D. Diz-Lopes, Eng. T. Coutinhond Mrs. C. Abraão, for their assistance with sample collec-ion and preparation; and the Board and staff of VTH-UTADnd cat owners, for their co-operation.

eferences

yllon, T., Tesouro, M.A., Amusategui, I., Villaescusa, A., Rodriguez-Franco, F., Sainz, A., 2008. Serologic and molecular evaluation ofLeishmania infantum in cats from Central Spain. Ann. N. Y. Acad. Sci.1149, 361–364.

aneth, G., Koutinas, A., Solano-Gallego, L., Bourdeau, P., Ferrer, L., 2008.Canine leishmaniosis – new concepts and insights on an expandingzoonosis: part one. Trends Parasitol. 24, 325–330.

ampino, L., Pratlong, F., Abranches, P., Rioux, J.-A., Santos-Gomes, G.,Alves-Pires, C., Cortes, S., Ramada, J., Cristóvão, J.M., Afonso, M.O.,Dedet, J.P., 2006. Leishmaniasis in Portugal: enzyme polymorphism ofLeishmania infantum based on the identification of 213 strains. Trop.Med. Int. Health 11, 1708–1714.

ardoso, L., Santos, H., Cordeiro-da-Silva, A., Pratlong, F., Dedet, J.-P.,Rodrigues, M., 2002. Leishmania infantum MON-98: infection in a dogfrom Alto Douro, Portugal. Acta Trop. 83, 83–85.

ardoso, L., Rodrigues, M., Santos, H., Schoone, G.J., Carreta, P., Varejão,E., van Benthem, B., Afonso, M.O., Alves-Pires, C., Semião-Santos, S.J.,Rodrigues, J., Schallig, H.D.F.H., 2004a. Sero-epidemiological study ofcanine Leishmania spp. in the municipality of Alijó (Alto Douro, Por-tugal). Vet. Parasitol. 121, 21–32.

ardoso, L., Schallig, H.D.F.H., Neto, F., Koon, N., Rodrigues, M., 2004b. Sero-logical survey of Leishmania infection in dogs from the municipalityof Peso da Régua (Alto Douro, Portugal) using the direct agglutinationtest (DAT) and fast agglutination screening test (FAST). Acta Trop. 91,95–100.

esjeux, P., 2001. Worldwide increasing risk factors for leishmaniasis.Med. Microbiol. Immunol. 190, 77–79.

iakou, A., Papadopoulos, E., Lazarides, K., 2009. Specific anti-Leishmania

spp. antibodies in stray cats in Greece. J. Feline Med. Surg. 11,728–730.

uarte, A., Castro, I., Fonseca, I.P., Almeida, V., Carvalho, L.M., Meireles, J.,Fazendeiro, M.I., Tavares, L., Vaz, Y., 2010. Survey of infectious and par-asitic diseases in stray cats at the Lisbon Metropolitan Area, Portugal.J. Feline Med. Surg. 12, 441–446.

itology 174 (2010) 37–42 41

Durão, J.F.C., Rebelo, E., Peleteiro, M.C., Correia, J.J., Simões, G., 1994.Primeiro caso de leishmaniose em gato doméstico (Felis catusdomesticus) detectado em Portugal (Concelho de Sesimbra): nota pre-liminar. Rev. Port. Cienc. Vet. 89, 140–144.

Hervás, J., de Lara, C.-M., Sánchez-Isarria, F., Pellicer, F.A., Carrasco, L.,Castillo, J.A., Gómez-Villamandos, J.C., 1999. Two cases of feline vis-ceral and cutaneous leishmaniosis in Spain. J. Feline Med. Surg. 1,101–105.

Lopes, A.P., Cardoso, L., Rodrigues, M., 2008. Serological survey ofToxoplasma gondii infection in domestic cats from northeastern Por-tugal. Vet. Parasitol. 155, 184–189.

Maia, C., Nunes, M., Campino, L., 2008. Importance of cats inzoonotic leishmaniasis in Portugal. Vector Borne Zoonotic Dis. 8,555–559.

Mancianti, F., 2004. Leishmaniosi felina: quale ruolo epidemiologico?Parasitologia 46, 203–206.

Marcos, R., Santos, M., Malhão, F., Pereira, R., Fernandes, A.C., Montenegro,L., Roccabianca, P., 2009. Pancytopenia in a cat with visceral leishma-niasis. Vet. Clin. Pathol. 38, 201–205.

Maroli, M., Pennisi, M.G., Di Muccio, T., Khoury, C., Gradoni, L., Gramic-cia, M., 2007. Infection of sandflies by a cat naturally infected withLeishmania infantum. Vet. Parasitol. 145, 357–360.

Martín-Sánchez, J., Acedo, C., Munoz-Pérez, M., Pesson, B., Marchal, O.,Morillas-Márquez, F., 2007. Infection by Leishmania infantum in cats:epidemiological study in Spain. Vet. Parasitol. 145, 267–273.

Miró, G., Cardoso, L., Pennisi, M.G., Oliva, G., Baneth, G., 2008. Canine leish-maniosis – new concepts and insights on an expanding zoonosis: parttwo. Trends Parasitol. 24, 371–377.

Molina, R., Amela, C., Nieto, J., San-Andrés, M., González, F., Castillo, J.A.,Lucientes, J., Alvar, J., 1994. Infectivity of dogs naturally infected withLeishmania infantum to colonized Phlebotomus perniciosus. Trans. R.Soc. Trop. Med. Hyg. 88, 491–493.

Nasereddin, A., Salant, H., Abdeen, Z., 2008. Feline leishmania-sis in Jerusalem: serological investigation. Vet. Parasitol. 158,364–369.

Ozon, C., Marty, P., Pratlong, F., Breton, C., Blein, M., Lelièvre, A., Haas, P.,1998. Disseminated feline leishmaniosis due to Leishmania infantumin Southern France. Vet. Parasitol. 75, 273–277.

Pennisi, M.G., 2002. A high prevalence of feline leishmaniasis insouthern Italy. In: Killick-Kendrick, R. (Ed.), Canine Leishmaniasis:Moving Towards a Solution. Intervet International, Boxmeer, pp.39–48.

Petrie, A., Watson, P., 1999. Statistics for Veterinary and Animal Science.Blackwell, Oxford, 243 pp.

Poli, A., Abramo, F., Barsotti, P., Leva, S., Gramiccia, M., Ludovisi, A., Man-cianti, F., 2002. Feline leishmaniosis due to Leishmania infantum inItaly. Vet. Parasitol. 106, 181–191.

Sanches, A., Cardoso, C., Pereira, A., Carvalho, J., 2009. Um caso de leish-maniose felina. In: Proceedings of the 18th APMVEAC Congress,Lisbon, Portugal.

Santarém, N., Silvestre, R., Cardoso, L., Schallig, H., Reed, S.G., Cordeiro-da-Silva, A., 2010. Application of an improved enzyme-linkedimmunosorbent assay method for serological diagnosis of canineleishmaniasis. J. Clin. Microbiol. 48, 1866–1874.

Santos, H.J.P., 1997. Contribuicão para o estudo da leishmaniose canina naregião do Alto Douro: estudo da prevalência no concelho de Tabuaco.University of Trás-os-Montes e Alto Douro, Vila Real, 91 pp.

Sarkari, B., Hatam, G.R., Adnani, S.J., Asgari, Q., 2009. Seroprevalence offeline leishmaniasis in areas of Iran where Leishmania infantum isendemic. Ann. Trop. Med. Parasitol. 103, 275–277.

Schallig, H.D.F.H., Schoone, G.J., Beijer, E.G.M., Kroon, C.C.M., Hommers,M., Özbel, Y., Özensoy, S., da Silva, E.S., Cardoso, L.M., da Silva, E.D.,2002. Development of a fast agglutination screening test (FAST) forthe detection of anti-Leishmania antibodies in dogs. Vet. Parasitol. 109,1–8.

Sherry, K., Miró, G., Trotta, M., Miranda, C., Montoya, A., Espinosa, C., Ribas,F., Furlanello, T., Solano-Gallego, L., 2010. A serological and molecularstudy of Leishmania infantum infection in cats from the Island of Ibiza(Spain). Vector Borne Zoonotic Dis, 2010 Aug 30. [Epub ahead of print].

Simões-Mattos, L., Bevilaqua, C.M.L., Mattos, M.R.F., Pompeu, M.M.L., 2004.Feline Leishmaniasis: uncommon or unknown? Rev. Port. Cienc. Vet.99, 79–87.

Solano-Gallego, L., Morell, P., Arboix, M., Alberola, J., Ferrer, L., 2001. Preva-

lence of Leishmania infantum infection in dogs living in an area ofcanine leishmaniasis endemicity using PCR on several tissues andserology. J. Clin. Microbiol. 39, 560–563.

Solano-Gallego, L., Rodríguez-Cortés, A., Iniesta, L., Quintana, J.,Pastor, J., Espada, Y., Portús, M., Alberola, J., 2007. Cross-sectional serosurvey of feline leishmaniasis in ecoregions around

ry Paras

1990–1998. WHO/LEISH/200.42. World Health Organization, Geneva,

42 L. Cardoso et al. / Veterina

the Northwestern Mediterranean. Am. J. Trop. Med. Hyg. 76,676–680.

Tabar, M.D., Altet, L., Francino, O., Sánchez, A., Ferrer, L., Roura, X., 2008.Vector-borne infections in cats: molecular study in Barcelona area(Spain). Vet. Parasitol. 151, 332–336.

Vita, S., Santori, D., Aguzzi, I., Petrotta, E., Luciani, A., 2005. Feline leish-maniasis and ehrlichiosis: serological investigation in Abruzzo region.Vet. Res. Commun. 29 (Suppl. 2), 319–321.

itology 174 (2010) 37–42

WHO, 2000. Leishmania/HIV Co-infection – Southwestern Europe

12 pp.Zárate-Ramos, J.J., Arbea-Sarasa, I., Gómez-Ochoa, P., Castillo-Hernández,

J.A., García-Salinas, M.J., Morales-Amella, M.J., 2002. Serological evi-dence of leishmaniasis in cats in Aragon, Spain. In: Proceedings of the27th WSAVA Congress, Granada, Spain.