Seroepidemiological survey of Leishmania infantum infection in dogs from northeastern Portugal

Acta Tropica 120 (2011) 82–87

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Seroepidemiological survey of Leishmania infantum infection in dogs from northeastern Portugal Susana Sousa a , Ana Patricia Lopes c , Luís Cardoso a,c , Ricardo Silvestre a , Henk Schallig d , Steven G. Reed e , Anabela Cordeiro da Silva a,b,∗ a

Parasite Disease Group, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal c Departamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal d KIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute), KIT Biomedical Research, Amsterdam, The Netherlands e Infectious Disease Research Institute, Seattle, WA, USA b

a r t i c l e

i n f o

Article history: Received 14 February 2011 Received in revised form 8 June 2011 Accepted 14 June 2011 Available online 2 July 2011 Keywords: Leishmania infantum Dogs Seroprevalence DAT ELISA

a b s t r a c t Northeastern Portugal is a region where canine leishmaniasis (CanL) is endemic. In this study, a seroepidemiological survey was conducted in 654 dogs from that geographical area. Serum samples were evaluated by the direct agglutination test (DAT) and also by enzyme-linked immunosorbent assay (ELISA) using five different defined antigens. Seroprevalence of infection was 21.3% based on the assumption that seropositive animals were positive for at least three tests. A high degree of agreement was found between DAT and LAM-ELISA (89%; kappa value [] = 0.67). A statistically significant difference (p < 0.05) of seropositivity was found between adult (23.4%) and juvenile dogs (12.2%), apparently healthy (14.8%) and sick dogs (40.2%), vaccinated (19.7%) and non-vaccinated (41.2%) animals, seropositive (26.9%) and seronegative (18.0%) for Toxoplasma gondii, living in rural (18.5%) or urban (32.6%) areas, and between animals living exclusively outdoors (18.2%) and those living in a mixed habitat (27.5%). Risk factors for canine Leishmania infection, as defined by multiple logistic regression analysis, were of clinical status (odds ratio [OR] = 3.1) and Toxoplasma infection (OR = 1.5). © 2011 Elsevier B.V. All rights reserved.

1. Introduction Leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania and is the most important vector-borne disease after malaria and sleeping sickness (Solano-Gallego et al., 2009). The Mediterranean basin, the Indian subcontinent and South America are geographic areas where Leishmania infection is endemic (Bettini and Gradoni, 1986; Alvar et al., 2004). According to the World Health Organization (WHO), the public health impact of leishmaniasis worldwide has been grossly underestimated (Ready, 2010). In 2001, 350 million people were calculated to be at risk, with 12 million patients and 2 million new cases per year (Desjeux, 2001). About 2 million new cases of human leishmaniasis are considered to occur every year in the endemic regions (Ready, 2010). Dogs are considered the main reservoir of Leishmania infantum (syn. Leishmania chagasi), the agent of zoonotic visceral leishmaniasis (VL).

∗ Corresponding author at: Parasite Disease Group, Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. Tel.: +351 222078923; fax: +351 222003977. E-mail addresses: susanasousa [email protected] (S. Sousa), [email protected] (A. Cordeiro da Silva). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.06.003

CanL is an emergent disease in several parts of the world, including non-endemic regions as consequence of the increasing number of travelling dogs (Menn et al., 2010). CanL is a systemic, chronic disease, ranging from asymptomatic subclinical to symptomatic infection. The clinical signs usually include lymphadenopathy, dermatitis, alopecia, cutaneous ulcerations, onychogriposis, lameness (abnormal locomotion), anorexia (poor appetite), weight loss, cachexia, ocular lesions, epistaxis, anaemia, diarrhoea and renal failure (Koutinas et al., 1999). Nevertheless, more than half of the infected animals are apparently healthy or asymptomatic (SolanoGallego et al., 2001), but are still capable of transmitting the parasite to the vector, the phlebotomine sand flies (Guarga et al., 2000). This represents an important veterinary and public health problem since it contributes to the maintenance of the Leishmania life cycle and transmission to humans. As a disease, the prevalence of CanL is frequently lower than 10% in endemic regions (Solano-Gallego et al., 2009), but the prevalence of canine Leishmania infection is thought to be considerably higher (Baneth et al., 2008). Current serological approaches to detect anti-Leishmania antibodies constitute a valuable alternative for early, rapid, and user-friendly diagnostic tests for both human and canine Leishmania infection. These serological methods, such as the direct

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agglutination test (DAT) and enzyme-linked immunosorbent assays (ELISA) have the advantage that they can be easily applied to a large number of serum samples with high levels of sensitivity and specificity (Scalone et al., 2002; Schallig et al., 2002). Therefore, the detection of Leishmania-specific antibodies is an important tool for the diagnosis of CanL. However, some Leishmania antigens currently used as diagnostic targets often perform sub optimally in detecting asymptomatic or subclinical infections. Total parasite extracts have been described as sensitive for the detection of subclinical and clinical canine infections, but with a lower specificity. The use of defined recombinant antigens often demonstrated high specificity and sensitivity depending on the antigen used. In addition, the use of Leishmania recombinant antigens is less prone to cross-reactivity, displaying lower false-positive reactions (Solano-Gallego et al., 2009). Some recombinant antigens have been already described as useful tools for the detection of infected dogs, such as rK39 (Goto et al., 2009) and L. infantum cytosolic tryparedoxin peroxidase (LicTXNPx) (Silvestre et al., 2008; Santarém et al., 2010). Recently, we have described a highly specific ELISA-based method that combining these two recombinant proteins with increasing specificity and sensitivity (Santarém et al., 2010). The objective of this study was to evaluate the seroprevalence of Leishmania infection in a large dog population from a region of northern Portugal where CanL is endemic. In addition, we calculated the association of several risk factors with Leishmania seroprevalence. 2. Materials and methods 2.1. Animals and serum samples Serum samples were randomly obtained from 654 domestic dogs of northeastern Portugal, in the years 2008 and 2009. Information for each sampled animal was registered on 11 independent variables: gender, sexual status (intact or neutered), breed (pure or mixed-breed), age group (juveniles up to and adults older than 12 months), clinical condition (apparently healthy or sick), vaccination, deworming, serology for Toxoplasma gondii, life in urban or rural environments, type of housing (indoor, outdoor or mixed), and whether or not they were hunting dogs. After owners’ informed consent, blood samples were collected from the cephalic or jugular vein into tubes without anticoagulant. Following centrifugation of clotted blood at 2,500 rpm for 10 min, serum aliquots were stored at −20 ◦ C. The 654 dog sera were tested for antibodies to Leishmania by means of DAT and ELISA with five different antigens describe below. Serological assessment of antibodies to T. gondii was carried out with ToxoScreen DA® (bioMérieux, Lyon, France), as described by Lopes et al. (2011).

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trifuged at 13,000 × g for 30 min at 4 ◦ C to separate the nuclei and the supernatant taken as the parasite extract, aliquoted and stored at −80 ◦ C. Recombinant proteins, LicTXNPx and LimTXNPx were prepared as described by Cordeiro-da-Silva et al. (2003). These two proteins containing a six-histidine residue at its N-terminal were purified by affinity chromatography on a Ni-NTA column (Qiagen). All antigens were analysed on 12% polyacrylamide gels and visualized by staining with Coomassie blue. The protein content of each antigen preparation was determined by the Lowry assay. 2.3. ELISA protocol ELISA was performed as previously described (Silvestre et al., 2008). Briefly, 96 well-plates (Greiner) were coated with 10 ␮g/ml of SPLA and 5 ␮g/ml of LicTXNPx, LimTXNPx or rK39 diluted in carbonate/bicarbonate buffer 0.05 M pH 9.6. Wells were blocked with PBS/milk 5% for 1 h at 37◦ C. Sera were diluted to 1/1,500 in PBS/Tween 0.05% and incubated for 30 min at 37◦ C. Anti-dog IgG conjugated with horseradish peroxidase diluted to 1/5,000 was used as secondary antibody. Reaction was developed with ophenylenediamine (OPD) for 10 min. Reaction was stopped with HCl 3 M and absorbance measured at 492 nm. ELISA cut-off values were previously defined based on the ROC curve defined using the reactivity of sera from dogs negative to Leishmania, and the reactivity of positive sera (Santarém et al., 2010). Serum samples with OD higher than 0.156, 0.146, 0.094, 0.154 and 0.149 were considered positive for SPLA, LicTXNPx, rK39, LimTXNPx and LAM, respectively. 2.4. DAT The DAT for titration of antibodies specific to Leishmania followed the general procedures described by Schallig et al. (2002), using a standard freeze-dried antigen at a concentration of 5 × 107 promastigotes/ml (KIT Biomedical Research, Amsterdam, The Netherlands). Briefly, the serum samples were diluted in 0.9% NaCl saline containing 1.56% ␤-mercaptoethanol. Twofold dilution series were made ranging from 1:100 to 1:102,400 in V-shaped micro titre plates (Greiner, Germany) and incubated for 1 h at 37 ◦ C. Fifty microlitres of reconstituted DAT antigen was subsequently added to each well containing 50 ␮l of diluted serum. 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 clearly visible after 18 h incubation at room temperature. A cut-off titre of 400 has been chosen to maximize sensitivity and specificity of the serodiagnosis (Schallig et al., 2002). Positive controls for the DAT consisted of serum samples from dogs with leishmaniasis. DAT titres in these samples ranged between 51,200 and ≥102,400. Sera from dogs living in areas where leishmaniasis is not endemic were used as negative controls. 2.5. Statistical analysis

2.2. Antigens for ELISA Five antigens were used for ELISA assays: soluble promastigote Leishmania antigens (SPLA); L. infantum mithocondrial tryparedoxin peroxidase (LimTXNPx); Leishmania antigens mixture (LAM); L. infantum cytosolic tryparedoxin peroxidase (LicTXNPx); rK39. SPLA was prepared as previously described (Cordeiro-da-Silva et al., 2003). LAM was previously described as the combination of 4 ␮g/ml of rK39 and 1 ␮g/ml of LicTXNPx (Santarém et al., 2010). Briefly, parasites were washed three times in a phosphate-buffered saline (PBS), pH 7.2 (3,000 × g, 10 min, 4 ◦ C). The pellet was resuspended in PBS containing 2 mM PMSF protease inhibitor and the mixture was submitted to 10 freezing (at −80 ◦ C)–thawing water bath (at 37 ◦ C) cycles and sonication (Vibra Cell, Sonics & Materials, Inc., Danbury, CT) for complete rupture of the parasites. The mixture was cen-

The observed agreements between DAT and ELISA results were calculated. Kappa value () expressed the agreement beyond chance. Seroprevalence values of anti-Leishmania antibodies for each of the 11 categorical variables were statistically compared using the Chi-squared or Fisher’s exact tests. Confidence limits for the proportions were established by the exact binomial test with 95% confidence intervals (CI). Variables with a significant difference between groups (probability [p] value < 0.05) were selected for multiple logistic regression analysis to identify risk factors for seroprevalence, calculating odds ratios (OR) and their 95% CI (Altman, 1991). Sensitivity, specificity, positive predictive value and negative predictive value were calculated for each one of the tests by using the formulas “sensitivity = TP/(TP + FN) × 100%”, “specificity = TN/(TN + FP) × 100%”, “pos-

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itive predictive value = TP/(TP + FP) × 100%” and “negative predictive value = TN/(TN + FN) × 100%”, where TN is the number of true negatives, TP is the number of true positives, FN is the number of false negatives and FP is the number of false positives (Altman, 1991). Differences of OD (ELISA) and titre (DAT) between apparently healthy and sick dogs were analysed with the Mann–Whitney U test. 3. Results For the current seroepidemiological survey we have randomly collected 654 serum samples from domestic dogs of northeastern Portugal. According to DAT results, 137 out of the 654 sera were found positive (20.9%). The DAT titres ranged from 400 to 102,400. Two-hundred and seventeen serum samples (33.2%) were positive by SPLA ELISA. The recombinant proteins LicTXNPx and rK39 detected 166 positive (25.4%) and 141 positive (21.6%) out of 654 serum samples, respectively. ELISA using these two proteins, LAM-ELISA, was positive for 128 out of 654 serum samples (19.6%) (Fig. 1A–F) When comparing DAT and ELISA results, the higher agreement was observed between DAT and LAM-ELISA results with an agreement of 89% and a  value of 0.65, which represents a substantial agreement beyond chance (Table 1). The lowest proportion of agreement was observed between DAT and LicTXNPx ELISA (76%), with a  value of 0.32, which represents a fair agreement. In order to determine seroprevalence, we defined that seropositivity for three or more tests were considered Leishmania positive. Based on that assumption, we detected a seroprevalence of 21.3% (139/654 samples) in our study population. Seroprevalence was significantly higher for adult dogs (23.4%) when compared with juveniles (12.2%) (p = 0.008) (Table 2). Clinically healthy dogs had a seroprevalence of 14.8%, which was significantly lower (p < 0.001) than sick dogs that presented a seroprevalence of 40.2%. Vaccinated dogs had a significantly different seroprevalence (19.7%) compared with non-vaccinated animals (41.2%) (p < 0.001). Dogs living in an urban environment had a significantly higher seroprevalence (32.6%) than the ones living in rural areas (18.5%) (p < 0.001). Seroprevalence was higher for animals living outdoors but with indoor access (27.5%) than for animals living exclusively outdoors (18.2%).

Table 1 Comparison between DAT and ELISA results. ELISAa

SPLAc + − LicTXNPxd + − rK39 + − LimTXNPxe + − LAMf + − a b c d e f

Observed agreement

 value

101 416

81%

0.54

72 65

94 423

76%

0.32

100 37

43 474

88%

0.64

30 107

41 476

77%

0.17

95 42

32 485

89%

0.65

DATb +

−

116 21

Enzyme-linked immunosorbent assay. Direct agglutination test. Soluble promastigotes Leishmania antigens. L. infantum cytosolic tryparedoxin peroxidase. L. infantum mithocondrial tryparedoxin peroxidase. Leishmania antigens mixture.

However, dogs living exclusively indoors were all seronegative. Seropositive dogs for T. gondii had a significant (p = 0.007) different L. infantum seroprevalence (26.9%) compared to T. gondii seronegative animals (18%). No significant differences were found between male and female animals (p = 0.660), neutered and intact dogs (p = 0.201), pure and mixed breed animals (p = 0.146), dewormed and non-dewormed dogs (p = 0.701), or animals involved or not in hunting activities (p = 0.678). Multiple logistic regression analysis of variables with statistical significant differences identified clinical status (OR = 3.1) and Toxoplasma gondii infection (OR = 1.5) as risk factors for seroprevalence. Sick dogs and those seropositive for T. gondii had approximately 3-fold and 1.5-fold higher risks of being seropositive to Leishmania than healthy animals or T. gondii seronegative dogs, respectively.

Fig. 1. Levels of IgG antibodies against SPLA (A), LicTXNPx (B), rK39 (C), LimTXNPx (D) and LAM (E) and all five antigens in dog sera by ELISA. Results are expressed as the optical densities at 492 nm. Each dot represents an individual dog serum. Dotted lines represent cut-off values.

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Table 2 Seroprevalence of Leishmania infection in dogs from northeastern Portugal according to 11 independent variables. Dogs tested (n)

Leishmania positive (n)

Seroprevalence (%)

26.5 73.5

35 105

20.2 21.8 p = 0.660

14.5–27.0 0.2–25.8

621 33

95.0 5.0

130 10

20.9 30.3 p = 0.201

17.8–24.3 15.6–48.7

Breed Pure Mixed

390 264

59.6 40.4

76 64

19.5 24.2 p = 0.146

15.7–23.8 19.2–29.9

Age group Juvenile Adult

115 539

17.6 82.4

14 126

12.2 23.4 p = 0.008

6.8–19.6 19.9–27.2

Clinical condition Apparently healthy Sick

485 169

74.2 25.8

72 68

14,8 40,2 p < 0.001

11.8–18.3 32.8–48.0

Vaccination No Yes

51 603

7.8 92.2

21 119

41.2 19.7 p < 0.001

27.6–55.8 16.6–23.1

Deworming No Yes

220 434

33.6 66.4

49 91

22.3 21.0 p = 0.701

16.9–28.3 17.2–25.1

T. gondii Seronegative Seropositive

405 249

61.9 38.1

73 67

18.0 26.9 p = 0.007

14.4–22.1 21.5–32.9

Environment Rural Urban

519 135

79.4 20.6

96 44

18.5 32.6 p < 0.001

15.2–22.1 24.8–41.2

Housing Indoors Mixed Outdoors

7 236 411

1.1 36.1 62.8

0 65 75

0.0 27.5 18.2 p = 0.008

0.0–41.0 21.9–33.7 14.6–22.3

Hunting activity No Yes

534 120

81.7 18.3

116 24

21.7 20.0 p = 0.678

2.0–25.5 13.2–28.3

Total

654

140

21.4

Gender Female Male

173 481

Sexual status Intact Neutered

Relative distribution (%)

100

Assuming as seropositive those samples with three or more positive tests, relative sensitivity, specificity, positive and negative predictive values were: 97.1%, 84.2%, 62.7% and 99.1% for SPLA ELISA; 69.3%, 86.6%, 54,5% and 91,2% for LicTXNPx; 89.3%, 96.5%, 60.7% and 97.1% for rK39 ELISA; 26.4%, 93.4%, 31.4% and 82.3% for LimTXNPx ELISA, 82.9%, 97.9%, 58.9% and 95.4% for LAMELISA; 77.9%, 94.6%, 57.4% and 94.0% for DAT. Levels of antibodies to Leishmania as determined by OD (ELISA) or titre (DAT) were higher in sick dogs (data not shown) and significantly different for SPLA ELISA (p = 0.002), rK39 ELISA (p < 0.001), LimTXNPx ELISA (p = 0.046), LAM-ELISA (p < 0.001) and DAT (p < 0.001), but not for LicTXNPx (p = 0.459). 4. Discussion Northern Portugal is a geographical region where CanL is endemic. Dogs, specially the asymptomatically infected ones have an important role in maintaining the cycle of Leishmania, since these dogs are capable to transmit the protozoa to the vector and thus to disseminate the parasite to humans (Guarga et al., 2000). In the current study, we have determined the Leishmania seroprevalence using several defined antigens. Using as the seropositivity

95% CI

0.0–24.7

readout reactivity in three or more serological tests, we were able to determine a prevalence of 21.3% in the present study. Indeed, this value is in agreement with the seroprevalence value reported in previous studies conducted in two different municipalities (Peso da Régua and Alijó) of the same region. The seroprevalence of Leishmania infection in 294 dogs from Peso da Régua was found to be 20.4% (Cardoso et al., 2004b), and 18.7% in 1,540 dogs from Alijó (Cardoso et al., 2004a). Interestingly, in large seroepidemiological surveys recently performed, seroprevalence was found to be lower, ranging from 8.1% to 13% in Spain (Gálvez et al., 2010; Martín-Sánchez et al., 2009) and 8% in Italy (Maresca et al., 2009). Nevertheless, these seroprevalence differences found in distinct studies might not always reflect the real picture, since results vary according to the serological methods and the size of the studied sample. In the present study, the percentage of positive serum samples ranged from 19.6% (LAM-ELISA) to 33.2% (SPLA-ELISA). The highest agreement was observed between LAM-ELISA and DAT, which supports the usefulness of these antigens for the detection of Leishmania infected dogs. False positive results obtained with ELISA based on promastigote-derived antigens have already been described (Salotra et al., 2002), which might explain the

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high percentage of positive sera observed for SPLA-ELISA. In this study, provided sensitivity, specificity, positive and negative predictive values should be regarded as relative figures and interpreted with precaution as they were not calculated in consideration to a true gold standard but were instead derived from the assumption that samples with three or more positive results were seropositive. A significant difference was observed between juveniles and adult dogs, probably related to a cumulative exposure of adult dogs to transmission by vectors. Other authors (Cardoso et al., 2010) have also found a significant difference for age group in Leishmania infection in cats. The group comprising sick animals may include dogs with clinical signs of leishmaniasis, which can explain the higher seroprevalence found in this group. A significant difference was also found between vaccinated and non-vaccinated animals. Most of these dogs were vaccinated against rabies. In general, non-vaccinated dogs receive little or no health care, including treatment with phlebotomine sand fly insecticides with repellent effect, which may explain the significantly higher seroprevalence among these animals. Dogs living in an urban environment had a significantly higher seroprevalence. Since European sand flies are mainly found in rural areas, it should be expected to find a higher seroprevalence in rural areas. However, in urban areas, gardens may favor proliferation of sand flies, which combined with a higher number of dogs, may facilitate transmission. In fact, increase in vector density is correlated with an increase of CanL (Ximenes et al., 2006). This finding may suggest the growing expansion and urbanization of the disease in Portugal. The abundance of sand flies in urban areas can reflect the degree of adaptation of the vector to new favorable environments. The significant difference observed between dogs living exclusively outdoors and dogs living in mixed habitats (outdoors and indoors) is also difficult to explain. Apparently, indoor access does not protect animals from infection, since the highest seroprevalence was found for dogs living in mixed habitats. All animals living exclusively indoors were seronegative, although very few animals were included in this group. Dogs seropositive for T. gondii had a significantly higher seroprevalence compared with dogs seronegative for Toxoplasma. The fact that seropositivity to T. gondii was the double in adults compared with juvenile dogs (data not shown) and that 82.4% of the dogs that were adult, may contribute to explain the difference in the seroprevalence to Leishmania infection according to T. gondii positivity. Toxoplasma gondii infection was identified as a risk factor for canine Leishmania infection. Clinical status was also identified as a risk factor. Age and geographical zone have previously been identified as risk factors for canine Leishmania infection (Cardoso et al., 2004a). Several serological tests are available to detect anti-Leishmania antibodies. DAT and ELISA are two of the serological tests most used in sero-epidemiological studies, since they allow the specific evaluation of a large number of serum samples (Scalone et al., 2002; Schallig et al., 2002). DAT has been a reference test in the majority of seroprevalence studies and is very suitable for the serodiagnosis of canine Leishmania infection (Schallig et al., 2002). The most common antigen used in ELISA is the one derived from promastigotes stages of Leishmania. In the present study, ELISA was also performed using the recombinant proteins LicTXNPx and rK39 as antigens. Previous studies showed that LicTXNPx based ELISA and rK39 based ELISA had specificity higher than 89% and sensitivity higher than 75% (Scalone et al., 2002; Zijlstra et al., 1998; Santarém et al., 2010). LAM-ELISA performed better than LicTXNPx based ELISA and rK39 based ELISA, both in symptomatic and asymptomatic dogs and having higher specificity (Santarém et al., 2010). LAM-ELISA is a simple, rapid and sensitivity test that together with DAT may be a valuable tool for screening canine Leishmania infection.

Acknowledgements Authors thank dog owners for their cooperation, and staff at the University of Trás-os-Montes e Alto Douro (Parasitology Lab and Veterinary Teaching Hospital) and at Dr. Duarte Diz-Lopes Veterinary Clinic, for assistance with sample collection. SS was supported by fellowship from FCT and FEDER codes SFRH/BPD/65214/2009. RS was supported by FCT Program Ciência 2008. The work was supported by FCT and FEDER grant n◦ PTDC/CVT/110732/2009.

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