Veterinary Immunology and Immunopathology 117 (2007) 3541 www.elsevier.

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Anti-Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs
L. Cardoso a,*, H.D.F.H. Schallig b, A. Cordeiro-da-Silva c, M. Cabral c, J.M. Alunda d, M. Rodrigues a
Department of Veterinary Sciences, CECAV, University of Tras-os-Montes e Alto Douro, P.O. Box 1013, 5001-801 Vila Real, Portugal b KIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute), KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands c Faculdade de Farmacia e IBMC Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Anbal Cunha 164, 4050-047 Porto, Portugal d Department of Animal Health, Veterinary Faculty, University Complutense of Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain Received 18 July 2006; received in revised form 29 December 2006; accepted 24 January 2007
a

Abstract Canine infections with Leishmania infantum represent a considerable veterinary medical and public health problem. In this study, immunoglobulin G1 (IgG1) and IgG2 specic humoral responses were measured and compared with the delayed type hypersensitivity (DTH) cellular response to a leishmanin, in three groups of dogs clinically and serologically characterised as: (I) asymptomatic and direct agglutination test (DAT)-seronegative; (II) asymptomatic and DAT-seropositive; (III) DAT-seropositive and symptomatic. IgG2 was regarded as a marker of disease, since signicantly higher levels of this subclass were recorded in the symptomatic dogs. In contrast, the IgG1 response could not be related to clinically relevant infection. A high correlation was observed between IgG2 level and DAT titre; the correlations between IgG1 and IgG2 levels, and between IgG1 level and DAT titre were lower. This may indicate that IgG2 is the main subclass in the specic humoral response which is detected by the DAT. A reduced IgG2 response, albeit not signicantly different, was recorded among dogs with clear cellular immune responses detected by a DTH positive reaction. Furthermore, no correlations were observed between cellular response measured by DTH and humoral responses quantied by DAT titre or IgG1 and IgG2 levels. Combining serology and DTH skin test is a practical procedure to assess anti-Leishmania immune responses in dogs. # 2007 Elsevier B.V. All rights reserved.
Keywords: Direct agglutination test; Dog; IgG1; IgG2; Leishmania infantum; Leishmanin skin test

1. Introduction Canine leishmaniosis (CanL) caused by Leishmania infantum (syn. L. chagasi) is endemic in approximately 50 countries in Europe, Africa, Asia, and the Americas (Alvar et al., 2004). Due to the zoonotic nature of the disease, dogs infected with the phlebotomine sand y-borne parasite represent a problem for both veterinary medicine and public health. CanL is a systemic, chronic, and even fatal

Abbreviations: CanL, canine leishmaniosis; DAT, direct agglutination test; DTH, delayed-type hypersensitivity; LST, leishmanin skin test * Corresponding author at: Departamento de Ciencias Veterinarias, Universidade de Tras-os-Montes e Alto Douro, Apartado 1013, 5001801 Vila Real, Portugal. Tel.: +351 259 350 458; fax: +351 259 350 629. E-mail address: lcardoso@utad.pt (L. Cardoso). 0165-2427/$ see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.01.014

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condition characterized by lymphadenopathy, dermatitis, skin ulceration, onychogriposis, lameness, anorexia, weight loss, cachexia, conjunctivitis, epistaxis, anaemia, and renal dysfunction (Koutinas et al., 1999). However, a large majority of the infected animals does not develop clinical signs, remaining asymptomatic for variable periods of time (Fisa et al., 1999) or even sometimes for life. L. infantum infections in dogs constitute a spectrum in which clinical disease represents one pole and asymptomatic subclinical infection the other one. Resistance against the parasite is associated with the development of protective cell-mediated immunity, whereas susceptibility is associated with high levels of antibodies (Cabral et al., 1992; Pinelli et al., 1994). Immunological methods are useful to diagnose canine Leishmania infections and may also be used to characterize host immune responses. Detection of anti-Leishmania specic antibodies in sera remains an important diagnostic tool, and ELISA and direct agglutination test (DAT) have proved to be very suitable for the evaluation of large numbers of canine serum samples (Harith et al., 1989; Scalone et al., 2002; Schallig et al., 2002). On the other hand, the leishmanin skin test (LST) is still the method of choice to assess antiLeishmania specic delayed-type hypersensitivity (DTH) cellular responses in dogs, particularly under eld conditions (Cardoso et al., 1998; FernandezBellon et al., 2005). Levels of anti-Leishmania specic IgG subclasses in dogs have been suggested as a marker for the susceptibility and consequent clinical status of the infected animals. Since their rst investigation by Deplazes et al. (1995) in naturally infected dogs, several other authors have studied the production of specic IgG1 and IgG2, sometimes with opposite results (Bourdoiseau et al., 1997; Solano-Gallego et al., 2000, 2001; Leandro et al., 2001; Cordeiro-da Silva et al., 2003; Fernandez-Perez et al., 2003; Oliveira-Mendes et al., 2003; Quinnell et al., 2003a; Almeida et al., 2005; Iniesta et al., 2005; Reis et al., 2006). Here we report a study of specic IgG subclasses in a large cohort of dogs from Alto Douro, the most important endemic region of zoonotic leishmaniosis in Portugal (Abranches et al., 1993; Cardoso et al., 2004). The main research objectives were to investigate levels of anti-Leishmania IgG1 and IgG2 subclasses and their relationship with humoral (DAT) and cellular (DTH) immune responses in dogs with different clinical status.

2. Materials and methods 2.1. Animals and samples The study comprised 188 dogs of various breeds and ages from the zoonotic leishmaniosis endemic region of Alto Douro (northern Portugal). The animals were clinically classied into three groups. Group I: asymptomatic and DAT-negative animals (n = 94); group II: asymptomatic and DAT-positive animals (n = 22); group III: DAT-positive and symptomatic animals (n = 72). The animals from groups I and II were subjected to inspection during eld surveys of canine Leishmania infection and those from group III were observed at the Veterinary Hospital of the University of Tras-os-Montes e Alto Douro (Vila Real, Portugal). Clinical examination was performed by expert veterinarians. Dogs from groups I and II had no clinical signs of disease, while those from group III were selected on the basis of presenting at least two clinical signs compatible with CanL, including: lymphadenopathy, alopecia, dermatitis, skin ulceration, keratoconjunctivitis, onychogryposis, lameness, epistaxis, anorexia and weight loss. Blood samples for serology were collected from the cephalic vein, processed and stored at 20 8C the same day. Prior to analysis, all sera were heat-inactivated (56 8C, 30 min); heat inactivation does not affect the results of the serological tests employed. Giemsa-stained smears of bone marrow aspirates of all animals included in the study were examined for the presence of amastigotes. 2.2. DAT DAT for the preliminary titration of specic antibodies used a standard freeze-dried antigen at a concentration of 5 107 promastigotes/ml (KIT Biomedical Research, Amsterdam, The Netherlands) and followed the general procedures described by Schallig et al. (2002). Results obtained with DAT are expressed as an antibody titre, i.e. the reciprocal of the highest dilution at which agglutination (large diffuse blue mats) is still visible after 18 h incubation at room temperature. A cutoff titre of 400 has been chosen to maximize sensitivity and specicity of the serodiagnosis (Schallig et al., 2002). 2.3. ELISA for IgG subclasses The quantication of specic IgG1 and IgG2 subclasses was carried out by an ELISA, with antigen concentration plus serum and conjugates dilutions determined by a checker-board titration (Voller, 1976). Microtitre plates (MaxiSorp Surface, Nunc, USA) were

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coated with 100 ml of soluble antigen obtained from sonicated promastigotes of a local L. infantum strain and adjusted to a protein concentration of 5 mg/ml in 0.05 M sodium carbonate buffer (pH 9.6). Plates were left overnight at 4 8C; blocked with a 5% (w/v) BSA solution in 0.05% (v/v) PBS-Tween 20 (PBS-T, pH 7.2) for 37 8C at 1 h and then washed three times with PBS-T. Canine sera were diluted at 1:1000 for IgG1 and 1:6000 for IgG2, in a solution of PBS-T with 1% BSA. One hundred microlitres of each serum dilution was added per well and incubated at 37 8C for 1 h. All the plates included a positive and a negative control sera. After three washings with PBS-T, horseradish peroxidase-conjugated goat anti-dog IgG1 and sheep anti-dog IgG2 (Bethyl Laboratories, USA) were used as secondary antibodies, respectively, at the dilutions of 1:1000 and 1:8000 in PBS-T. A volume of 100 ml per well of each conjugate dilution was incubated at 37 8C for 1 h, and the plates were then rewashed as above. The color reaction was developed with a solution of 0.01% (w/v) substrate 2,20 azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS, Sigma, USA) and 0.008% (v/v) hydrogen peroxide in 0.1 M of phosphate/citrate buffer (pH 4). One hundred microlitres of the solution was added per well and incubated at 37 8C for 30 min in the absence of light. OD were immediately read at 405 nm by using an automatic spectrophotometer (Titertek Multiskan Plus MK II, Flow Laboratories, Switzerland). OD values were converted into units (U) using serum from a DAT-seropositive symptomatic dog (titre ! 102,400) as reference and arbitrarily set at 100 U (Iniesta et al., 2005). All the samples were tested in triplicate and the results are presented as the average of those three determinations. Cut-off values were calculated as the mean plus 4 SD (Solano-Gallego et al., 2001) of 34 dogs from non-endemic areas, and established at 22.0 U for IgG1 and 19.3 U for IgG2. 2.4. LST After obtaining consent from the owners, it was possible to simultaneously examine 59 out of the 188 dogs by means of the LST to assess anti-Leishmania specic DTH cellular immune responses: 40 dogs were from group I, 15 from group II, and 4 from group III. The leishmanin consisted of 0.1 ml of a suspension containing 3 106 L. infantum promastigotes/ml in 0.5% (w/v) phenol/PBS and was intradermally injected in the abdominal wall. DTH skin reactions were measured 72 h later, by outlining the indurated border with a ballpoint pen. The largest diameter and its perpendicular diameter were measured. An average diameter of

induration !5 mm was considered as a positive result (Cardoso et al., 1998). A PPD (Statens Serum Institut, Denmark) and phenol/PBS were used as controls. 2.5. Data analysis Differences between independent groups were analysed with the MannWhitney U test, incorporating the Bonferroni correction (Petrie and Watson, 1999). Chi-square (x2) test was used to compare proportions. Association between variables was measured by the Spearmans rank correlation coefcient. Analyses were performed with SPSS 10.0 software for Windows, with a p value <0.05 as statistically signicant. 3. Results The set of results of DAT analysis and parasitological examination of the 188 dogs is presented in Table 1. DAT titres were signicantly higher ( p < 0.003) in the symptomatic group III than in the asymptomatic but DAT-seropositive group II. Parasites could not be detected in Giemsa-stained bone marrow slides of dogs in group I. Only two dogs, with relatively high DAT titres, were found parasitologically positive in the asymptomatic group II. In contrast, a signicant higher number of dogs were parasitologically positive in group III ( p < 0.001). The levels of anti-Leishmania specic IgG1 and IgG2 subclasses and IgG2/IgG1 ratios in groups I, II and
Table 1 Distribution of direct agglutination test (DAT) titres and parasitological examination results in 188 dogs classied into three groups on the basis of clinical examination and anti-Leishmania specic serology by DAT DAT titre <100 100 200 400 800 1600 3200 6400 12,800 25,600 51,200 !102,400 Total Group I (n) 82 (0) 9 (0) 3 (0) 94 (0) Group II (n) 2 1 2 3 2 5 5 2 Group III (n) 1 1 4 1 2 18 45

(0) (0) (0) (0) (0) (0) (1) (1)

(0) (0)

(0) (1) (2) (13) (36)

22 (2)

72 (52)

Group I: asymptomatic and DAT-negative; group II: asymptomatic and DAT-positive; group III: DAT-positive and symptomatic. DAT cutoff titre = 400. Numbers between parentheses represent animals found parasitologically positive by the examination of Giemsa-stained smears of bone marrow.

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L. Cardoso et al. / Veterinary Immunology and Immunopathology 117 (2007) 3541

were calculated for each group. Group I: 0.49 (IQR: 0.211.13); group II: 1.04 (IQR: 0.312.81); group III: 2.70 (IQR: 1.854.86). A higher correlation was observed between IgG2 level and DAT titre (r = 0.836; p < 0.001). Lower correlations were found between IgG1 and IgG2 levels (r = 0.663; p < 0.001), IgG2/IgG1 ratio and DAT titre (r = 0.641; p < 0.001), and IgG1 level and DAT titre (r = 0.577; p < 0.001). Signicant differences ( p < 0.003) were found between all the groups compared in pairs with respect to IgG2 level. On the other hand, differences in the levels of IgG1 were signicant between groups I and II

Fig. 1. Anti-Leishmania specic IgG1 and IgG2 levels (U) and IgG2/ IgG1 ratio in asymptomatic and DAT-negative dogs (group I, n = 94*), asymptomatic and DAT-positive dogs (group II, n = 22), and DATpositive and symptomatic dogs (group III, n = 72). Bars represent minimum and maximum values; boxes display median and interquartile range. Dotted lines represent cut-off values: 22.0 U for IgG1, and 19.3 U for IgG2.

III are presented in Fig. 1. Medians of IgG1 levels were 9.7 U for group I (interquartile range [IQR]: 6.117.0); 22.3 U for group II (IQR: 11.254.9); 35.2 U for group III (IQR: 15.160.1). Medians of IgG2 levels were as follows. Asymptomatic and DAT-negative dogs (group I): 4.3 U (IQR: 2.111.2); asymptomatic and DATpositive dogs (group II): 23.2 U (IQR: 7.564.0); DATpositive and symptomatic dogs (group III): 107.5 U (IQR: 79.0117.9). Medians of the IgG2/IgG1 ratios

Fig. 2. Anti-Leishmania specic DTH values, and IgG1 and IgG2 levels (U) in 47 LST-negative and 12 LST-positive dogs. Bars represent minimum and maximum values; boxes display median and interquartile range. Dotted lines represent cut-off values: 5 mm for LST, 22.0 U for IgG1, and 19.3 for IgG2.

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( p < 0.003) and groups I and III ( p < 0.003), but not between groups II and III ( p = 0.606). Differences in the IgG2/IgG1 ratio were signicant between groups I and III ( p < 0.003) and groups II and III ( p < 0.003), but not between groups I and II ( p = 0.096). Anti-Leishmania specic cellular immune responses were studied in a sub-population (n = 59) of the study cohort and found positive in 12 dogs (8 animals from group I and 4 from group II) and negative in 47 dogs (32 animals from group I, 11 from group II and 4 from group III). All four tested symptomatic dogs from group III had a negative LST result. Fig. 2 presents the DTH values of induration and/or erythema, and specic IgG1 and IgG2 levels in the 59 dogs that were assessed by LST. Medians of DTH values were 0 mm for the LSTnegative (IQR: 00) and 8.5 mm for the LST-positive animals (IQR: 7.311.4). The levels of IgG1 had medians of 15.2 U for the LST-negative (IQR: 9.230.3) and 20.1 U (IQR: 13.436.2) for the LST-positive dogs. Medians of IgG2 levels were 6.7 U for the LST-negative (IQR: 2.218.5) and 4.1 U for the LST-positive (IQR: 2.37.4). DTH values were signicantly higher in the LST-positive than in the LST-negative animals ( p < 0.001), but no signicant differences in the levels of IgG1 ( p = 0.221) and IgG2 ( p = 0.402) were found between LST-negative and LST-positive dogs. No correlation was observed between IgG1 and DTH (r = 0.146; p = 0.270), IgG2 and DTH (r = 0.116; p = 0.382) or DAT and DTH (r = 0.047; p = 0.726). Three dogs that were both LST and DAT positive had levels of IgG1 and IgG2 below their respective cut-off values. 4. Discussion Canine Leishmania infections constitute a clinical and immunological spectrum where susceptibility is associated with high levels of specic antibodies and changes in T cell responses, including absence of DTH to leishmanial antigens. An association of CD4+ Th2 cytokines IL-4 and IL-10 with symptomatic infections has been suggested, but it is still controversial whether these cytokines correlate with active CanL. On the other hand, CD4+ Th1 subsets producing IFN-g, IL-2 and TNFa are involved in resistance to the disease, as they have been found predominant in asymptomatic dogs. CD8+ cytotoxic T cells seem also to take part in the process of resistance to CanL. These cells are directly involved in the lysis of L. infantum infected macrophages from asymptomatic animals (reviewed by Barbieri, 2006). IgG1 and IgG2 subclasses have been suggested as more reliable markers of the clinical status in CanL than

total IgG (Deplazes et al., 1995). In the present study, IgG2 was regarded as a marker of active disease, since signicantly higher levels of this subclass were recorded in the symptomatic dogs (group III) in comparison with asymptomatic seropositive animals (group II). On the other hand, IgG1 response seemed to point only towards clinically indistinct Leishmania infection, since signicant differences were found between DAT-seronegative (group I) and seropositive dogs (groups II and III), but not between symptomatic and asymptomatic seropositive animals. Our ndings with regard to IgG2 agree with those of Leandro et al. (2001), but are contrary to what several other authors have suggested (Deplazes et al., 1995; Nieto et al., 1999; Iniesta et al., 2005). Furthermore, in a cohort of Brazilian dogs, Quinnell et al. (2003a) found that L. chagasi (syn. L. infantum) natural infection involved subclasses IgG1-4, with lower IgG2 values and IgG2/IgG1 ratios in the symptomatic animals. This apparent discrepancy of results can be due to the different strains/species of Leishmania parasites involved and/or to differences in the host breed and genetic background. In this way, a few other studies have demonstrated that susceptibility to CanL is linked to the polymorphism in canine NRAMP1 gene (Altet et al., 2002) and is also associated with MHC alleles (Quinnell et al., 2003b). A reduced albeit not signicantly different IgG2 response was recorded among dogs with cellular immune responses detected by a LST positive reaction. Solano-Gallego et al. (2000) and Leandro et al. (2001) did also not observe any association between IgG1 or IgG2 and anti-Leishmania specic cellular response as detected by LST or lymphocyte proiferation, respectively. On the other hand, high IgG2 values and a IgG1/ IgG2 ratio 1 were referred by Oliveira-Mendes et al. (2003) as being associated with a protective immune response and a positive LST in dogs vaccinated with the fucose-mannose ligand of L. donovani. One explanation for such difference from our study may be the fact that these authors analysed IgG1 and IgG2 subclasses specic for a dened antigen of L. donovani, whereas we used a whole soluble extract of L. infantum. In the present study, a high correlation was found between IgG2 level and DAT titre, strongly suggesting IgG2 as the main subclass of the anti-Leishmania specic humoral response which is detected by the DAT. To our knowledge, this is the rst reported comparison between DAT and IgG subclasses in dogs. Hereafter, it will also be important to further compare DAT titres and other specic IgG subclasses (Quinnell et al., 2003a,b) or even IgE and IgA classes (Almeida et al., 2005;

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L. Cardoso et al. / Veterinary Immunology and Immunopathology 117 (2007) 3541 identication of mutations in leishmaniasis-susceptible dogs. Infect. Immun. 70, 27632771. Alvar, J., Canavate, C., Molina, R., Moreno, J., Nieto, J., 2004. Canine leishmaniasis. Adv. Parasitol. 57, 188. Barbieri, C.L., 2006. Immunology of canine leishmaniasis. Parasite Immunol. 28, 329337. Bourdoiseau, G., Bonnefont, C., Hoareau, E., Boehringer, C., Stolle, T., Chabanne, L., 1997. Specic IgG1 and IgG2 antibody and lymphocyte subset levels in naturally Leishmania infantuminfected treated and untreated dogs. Vet. Immunol. Immunopathol. 59, 2130. Cabral, M., OGrady, J., Alexander, J., 1992. Demonstration of Leishmania specic cell mediated and humoral immunity in asymptomatic dogs. Parasite Immunol. 14, 531539. Cardoso, L., Neto, F., Sousa, J.C., Rodrigues, M., Cabral, M., 1998. Use of a leishmanin skin test in the detection of canine Leishmania-specic cellular immunity. Vet. Parasitol. 79, 213220. Cardoso, L., Rodrigues, M., Santos, H., Schoone, G.J., Carreta, P., Varejao, E., van Benthem, B., Afonso, M.O., Alves-Pires, C., Semiao-Santos, S.J., Rodrigues, J., Schallig, H.D.F.H., 2004. Sero-epidemiological study of canine Leishmania spp. infection in the municipality of Alijo (Alto Douro Portugal). Vet. Parasitol. 121, 2133. Cordeiro-da-Silva, A., Cardoso, L., Araujo, N., Castro, H., Tomas, A., Rodrigues, M., Cabral, M., Vergnes, B., Sereno, D., Ouaissi, A., 2003. Identication of antibodies to Leishmania silent information regulatory 2 (SIR2) protein homologue during canine natural infections: pathological implications. Immunol. Lett. 68, 155 162. Deplazes, P., Smith, N.C., Arnold, P., Lutz, H., Eckert, J., 1995. Specic IgG1 and IgG2 antibody responses of dogs to Leishmania infantum and other parasites. Parasite Immunol. 17, 451458. Fernandez-Bellon, H., Solano-Gallego, L., Rodrguez, A., Rutten, V.P.M.G., Hoek, A., Ramis, A., Alberola, J., Ferrer, L., 2005. Comparison of three assays for the evaluation of specic cellular immunity to Leishmania infantum in dogs. Vet. Immunol. Immunopathol. 107, 163169. Fernandez-Perez, F.J., Gomez-Munoz, M.T., Mendez, S., Alunda, J.M., 2003. Leishmania-specic lymphoproliferative responses ans IgG1/IgG2 immunodetection patterns by Western blot in asymptomatic, symptomatic and treated dogs. Acta Trop. 86, 8391. Fisa, R., Gallego, M., Castillejo, S., Aisa, M.J., Serra, T., Riera, C., Carrio, J., Gallego, J., Portus, M., 1999. Epidemiology of canine leishmaniosis in Catalonia (Spain). The example of the Priorat focus. Vet. Parasitol. 83, 8797. Gradoni, L., 2001. An update on antileishmanial vaccine candidates and prospects for a canine Leishmania vaccine. Vet. Parasitol. 100, 87103. Harith, A.E., Slappendel, R.J., Reiter, I., van Knapen, F., De Korte, P., Huigen, E., Kolk, A.H.J., 1989. Application of a direct agglutination test for detection of a specic anti-Leishmania antibodies in the canine reservoir. J. Clin. Microbiol. 27, 22522257. Iniesta, L., Fernandez-Barredo, S., Bulle, B., Gomez, M.T., Piarroux, R., Gallego, M., Alunda, J.M., Portus, M., 2002. Diagnostic techniques to detect cryptic leishmaniasis in dogs. Clin. Diagn. Lab. Immunol. 9, 11371141. Iniesta, L., Gallego, M., Portus, M., 2005. Immunoglobulin G and E responses in various stages of canine leishmaniosis. Vet. Immunol. Immunopathol. 103, 7781. Koutinas, A.F., Polizopoulou, Z.S., Saridomichelakis, M.N., Argyriadis, D., Fytianou, A., Plevraki, K.G., 1999. Clinical considera-

Iniesta et al., 2005; Reis et al., 2006). IgM antibodies are inactivated by b-mercapto-ethanol used in DAT protocol and thus not measured with this assay (Harith et al., 1989; Schallig et al., 2002). An effective vaccine for Leishmania should induce a stable and long-lasting Th1-type cellular response, and not a Th2-type or a mixed response (Gradoni, 2001). Prequisites for evaluating the immunogenicity of vaccines against canine Leishmania infection should include the assessment of specic humoral and cellular immune responses. IgG1 and IgG2 values may constitute markers of the protective response developed by vaccinated dogs and also represent a way to distinguish them from those naturally infected animals (Oliveira-Mendes et al., 2003). In the present study, several infected asymptomatic dogs had a positive cellular immune response that should be the basis for the protection against the development of disease (Cabral et al., 1992; Pinelli et al., 1994). The fact that three dogs with a DTH positive reaction and DAT-seropositive did not present detectable levels of IgG1 and IgG2, raises the hypothesis of other IgG subclasses related with the specic cellular response. These results are in the line of those obtained by Iniesta et al. (2002). However, complementary T cell proliferation and cytokine production studies need to be carried out in order to better characterise cellular immune responses. We conclude that the evaluation of anti-Leishmania cellular immunity is useful when considered in conjunction with antibody responses. Combining serology and the DTH skin test is a practical procedure to assess immune responses in dogs and to diagnose canine Leishmania infection. Acknowledgments The authors thank Dr. Birgit van Benthem (KIT Biomedical Research) and Prof. Jorge Colaco (Uni versity of Tras-os-Montes e Alto Douro) for their comments regarding the statistical analysis. References
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