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The use of Escherichia coli total antigens as a complementary approach to address seropositivity to Leishmania antigens in canine leishmaniosis

Published online by Cambridge University Press:  23 May 2017

CARLA LIMA
Affiliation:
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal
JOÃO RODRIGO MESQUITA
Affiliation:
Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Campus Agrário de Vairão, Vairão, Portugal Escola Superior Agrária, Instituto Politécnico de Viseu, Viseu, Portugal
HUGO BRANCAL
Affiliation:
Clínica Veterinária da Covilhã, Quinta das Ferreiras, Boidobra, Covilhã, Portugal Faculdade de Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal
THOMAS VAHLENKAMP
Affiliation:
Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany
ANA RAFAELA TEIXEIRA
Affiliation:
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
LUÍS CARDOSO
Affiliation:
Departamento de Ciências Veterinárias, Escola de Ciências Agrárias e Veterinárias, Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal
CÉLIA AMORIM
Affiliation:
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
NUNO SANTARÉM*
Affiliation:
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
ANABELA CORDEIRO DA SILVA*
Affiliation:
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal
*
*Corresponding author: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. E-mail: cordeiro@ff.up.pt or cordeiro@ibmc.up.pt and santarem@ibmc.up.pt
*Corresponding author: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. E-mail: cordeiro@ff.up.pt or cordeiro@ibmc.up.pt and santarem@ibmc.up.pt

Summary

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL – soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen – soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.

Type
Research Article
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
Copyright © Cambridge University Press 2017

INTRODUCTION

Canine leishmaniosis (CanL), caused by Leishmania infantum (syn. Leishmania chagasi), is endemic in the Mediterranean basin, South America and Southern Asia, being a major veterinary concern (Saridomichelakis, Reference Saridomichelakis2009; Ready, Reference Ready2010; Noli and Saridomichelakis, Reference Noli and Saridomichelakis2014; Wylie et al. Reference Wylie, Carbonell-Antonanzas, Aiassa, Dhollander, Zagmutt, Brodbelt and Solano-Gallego2014). These protozoa may also induce human visceral leishmaniasis, making CanL not just a veterinary problem but also a public health issue due to the zoonotic potential of the infection (Michalsky et al. Reference Michalsky, Rocha, da Rocha Lima, Franca-Silva, Pires, Oliveira, Pacheco, dos Santos, Barata, Romanha, Fortes-Dias and Dias2007; Sousa et al. Reference Sousa, Lopes, Cardoso, Silvestre, Schallig, Reed and Cordeiro da Silva2011; Noli and Saridomichelakis, Reference Noli and Saridomichelakis2014). CanL control is based on vaccination, transmission prevention by the insect vectors and also diseased animals treatment (Palatnik-de-Sousa, Reference Palatnik-de-Sousa2012; Courtenay et al. Reference Courtenay, Carson, Calvo-Bado, Garcez and Quinnell2014; Silva et al. Reference Silva, Starke-Buzetti, Alves-Martin, Paixão, Tenório and Lopes2014). The CanL diagnosis is based on clinical signs and demonstration of infection by direct visualization of parasites and/or genetic material in conjunction with quantitative serology (Michalsky et al. Reference Michalsky, Rocha, da Rocha Lima, Franca-Silva, Pires, Oliveira, Pacheco, dos Santos, Barata, Romanha, Fortes-Dias and Dias2007; Paltrinieri et al. Reference Paltrinieri, Solano-Gallego, Fondati, Lubas, Gradoni, Castagnaro, Crotti, Maroli, Oliva, Roura, Zatelli and Zini2010; Solano-Gallego et al. Reference Solano-Gallego, Miro, Koutinas, Cardoso, Pennisi, Ferrer, Bourdeau, Oliva and Baneth2011). Of paramount importance for disease management is the detection of dogs that have no leishmaniosis specific signs but are infected. It is well established that infected dogs without clinical signs are capable of perpetuating the zoonotic life cycle of the parasite (Molina et al. Reference Molina, Amela, Nieto, San-Andres, Gonzalez, Castillo, Lucientes and Alvar1994; Moreno and Alvar, Reference Moreno and Alvar2002). Therefore, low-accuracy serological assays can have significant impact in the global strategy to combat disease progression originating seropositivity levels that are not translatable into meaningful epidemiological data influencing policies associated with specific control measures. In fact, cumulative evidence point to a low specificity (Sp) of several serological assays that hamper the detection of Leishmania infection in dogs (Gomes et al. Reference Gomes, Paiva Cavalcanti, Lira, Abath and Alves2008; Morales-Yuste et al. Reference Morales-Yuste, Morillas-Marquez, Diaz-Saez, Baron-Lopez, Acedo-Sanchez and Martin-Sanchez2012; Arruda et al. Reference Arruda, Figueiredo, Marcelino, Barbosa, Werneck, Noronha and Romero2016; Elshafie et al. Reference Elshafie, Mullazehi and Ronnelid2016; Fraga et al. Reference Fraga, Pacheco, Borja, Tuy, Bastos, Solca Mda, Amorim and Veras2016). Moreover, detection of L. infantum vectors across central European countries like Switzerland, Germany and Hungary (Naucke et al. Reference Naucke, Menn, Massberg and Lorentz2008; Ready, Reference Ready2010), in conjunction with global warming, might open new areas to endemicity, through the adaptation of the vectors to new latitudes (Geisweid et al. Reference Geisweid, Weber, Sauter-Louis and Hartmann2013; Millan et al. Reference Millan, Ferroglio and Solano-Gallego2014). In fact, disease is advancing northwards, with reports of infected animals in traditionally non-endemic areas (Saari et al. Reference Saari, Rasi and Anttila2000; Dujardin et al. Reference Dujardin, Campino, Canavate, Dedet, Gradoni, Soteriadou, Mazeris, Ozbel and Boelaert2008; Ready, Reference Ready2010; Miro et al. Reference Miro, Checa, Montoya, Hernandez, Dado and Galvez2012; Carvalho et al. Reference Carvalho, Rangel, Ready and Vale2015; Maia and Cardoso, Reference Maia and Cardoso2015). Therefore, more than ever, it is essential to have adequate serosurveillance systems and tools to monitor and control infection progression into new regions.

Herein we evaluate serological response of an uncharacterized cohort of animals from distinct geographical backgrounds to two known Leishmania antigens [soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39)] to determine the adequacy of these antigens for serological surveys in non-endemic regions. To exclude any inter-laboratory variability, we used CanL-positive and negative cohorts of dogs to determine the cut-offs that are able to distinguish diseased from non-diseased animals for both antigens. The generated cut-offs were applied to the test cohort and levels of seropositivity compared. The unrelated soluble Escherichia coli antigen (SECA) was also used to address the Sp of the serological response to the Leishmania antigens. Finally, the ratios between the cut-off normalized responses to the antigens were evaluated for their capacity to predict disease in the control cohorts and then used as part of a scoring system that addresses seropositivity with clinical value.

MATERIALS AND METHODS

Canine sera

Group CanL+ (n = 29): sera from dogs living in geographical regions of Portugal where CanL is endemic, which were brought to a veterinary hospital, clinic or anti-rabies campaign and that presented at least two clinical signs compatible with the disease (viz. lymphadenomegaly, alopecia, dermatitis, skin ulceration, keratoconjunctivitis, onychogryphosis, lameness, epistaxis, anorexia and weight loss). These animals were also seropositive for anti-Leishmania antibodies by the direct agglutination test (DAT) (cut-off titre = 400) and/or positive for the presence of amastigotes in bone marrow or lymph node aspirates.

Group CanL− (n = 121): sera from dogs that visited a veterinary clinic in a Portuguese region considered to be non-endemic for CanL, Serra da Estrela mountain in central Portugal. All were seronegative by DAT (titre < 100).

Group Europe (n = 384): sera from dogs without clinical information, with unknown serological status, that visited veterinary clinics in several countries of Europe, namely Denmark (n = 50), France (n = 50), Germany (n = 47), Hungary (n = 47), the Netherlands (n = 42), Poland (n = 50), Portugal (n = 50) and the UK (n = 48).

Antigens

Three different antigens were used for enzyme-linked immunosorbent assay (ELISA): SPLA, SECA and Leishmania rK39. For SPLA, L. infantum promastigotes were obtained as previously described (Santarem et al. Reference Santarem, Silvestre, Cardoso, Schallig, Reed and Cordeiro-da-Silva2010). Parasites were washed three times with phosphate-buffered saline (PBS), pH 7·4 and centrifuged at 3500 g for 10 min at 4 °C. The pellet was resuspended in PBS containing 1 mm phenylmethylsulfonyl fluoride (PMSF) protease inhibitor and submitted to 10 freeze–thaw cycles for rupture of the parasites. This suspension was centrifuged at 13 000 g for 30 min at 4 °C and the supernatant was recovered, quantified by DC (detergent compatible)™ Protein Assay (BioRad, Munich, Germany), and stored at −80 °C in single aliquots.

The rK39 antigen obtained from Dr Steven Reed, from Infectious Disease Research Institute (Seattle, Washington, USA) was suspended in mili-Q water, quantified and stored at −80 °C in single-use aliquots.

For SECA, E. coli DH5-α strain was plated in an agar plate and incubated at 37 °C overnight (O/N). Then, a colony was transferred to 10 mL of Luria Bertani broth (Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C O/N. The next day, the optical density (OD) of the culture was measured at 600 nm (initial OD should be nearly 0·1) and left to grow for 3 h. The culture was then centrifuged at 4000 g for 5 min. The pellet was resuspended in 1 mL of PBS containing 1 mm of PMSF and submitted to 10 freeze–thaw cycles for rupture of the bacteria. The suspension was then centrifuged at 13 000 g for 30 min at 4 °C and the supernatant was recovered, quantified by DC (detergent compatible) Protein Assay (BioRad, Munich, Germany) and stored at −80 °C in single-use aliquots.

ELISA

Ninety-six-well flat-bottom microtitre plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 50 µL of 0·1 m carbonate buffer, pH = 9·6, with 10 µg mL−1 of either SPLA or SECA, or 1 µg mL−1 of rK39. Plates were incubated O/N at 4 °C and blocked with 200 µL of PBS low-fat milk (3%) at 37 °C for 1 h. Next, plates were washed with PBS-Tween 0·05% (PBS-T), and the sera, positive and negative controls, diluted at 1 : 1500 in PBS-T low-fat milk (1%), were dispensed in triplicate (100 µL per well) and incubated at 37 °C for 30 min. After a washing step, 100 µL per well of conjugate secondary anti-dog IgG antibody conjugated with horseradish peroxidase (Sigma, St. Louis, USA) – diluted at 1 : 1176·5, was added and the plates were incubated at 37 °C for 30 min. Plates were washed and incubated with 0·5 mg mL−1 of o-phenylenediamine dihydrochloride (Sigma, St. Louis, USA) for 10 min in dark. Reaction was stopped with 50 µL per well of HCl (3 m). Absorbance was read at 492 nm in an automatic reader (Synergy 2, BioTek Instruments, Winooski, USA).

All samples and antigens were assayed in at least two independent assays.

Statistical analysis

Receiver operating characteristic (ROC) curves were generated using sera from groups CanL+ and CanL−, using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. A 95% confidence interval for the area under the ROC curve was considered.

Cut-off values were inferred through these curves for each antigen [by choosing the best compromise between sensitivity (Se) and Sp associated with the ROC curve], and values of Se, Sp, false negatives (FN), false positives (FP), positive predictive value and negative predictive value were calculated for the samples for each group (Parikh et al. Reference Parikh, Mathai, Parikh, Chandra Sekhar and Thomas2008). Optical densities of each sample were normalized by division with the corresponding cut-off value (nSPLA, nrK39 and nSECA) and the logarithm of this ratio was applied for graphical representation [log(nSPLA), log(nrK39) and log(nSECA)]. The cut-off normalized values (n) were used to assess the ratio between the antigens: SPLA/SECA (ratio between nSPLA and nSECA), rK39/SECA (ratio between nrK39 and nSECA) and rK39/SPLA (ratio between nrK39 and nSPLA). A score evaluation method was established based on the cut-off values inferred from the ROC curves for the ratios and the single antigens. A binary score (1 or 0) was applied to represent seropositivity (arbitrary value of 1) or seronegativity (arbitrary value of 0) to the five serological parameters previously established and whose cut-off values was determined by ROC curve analysis: rK39, SPLA, rK39/SECA, SPLA/SECA and rK39/SPLA. Total cumulative score for each individual sample ranged from ‘0’ to ‘5’ (Fig. 1).

Fig. 1. Evaluation of seropositivity of each individual serum evaluated as a cumulative score. Each individual serum was ‘scored’ as nSPLA + nrK39 + nSECA + SPLA/SECA + rK39/SECA + rK39/SPLA using the following premise: each serology value above or equal to the cut-off values of nSPLA, nrK39, nSECA, SPLA/SECA, rK39/SECA and rK39/SPLA was transformed into a score of ‘1’ originating a cumulative score ranging from 0 to 5 to each individual sample.

RESULTS

ROC curve analysis was performed to define cut-off values for rK39 and SPLA using sera from dogs of CanL+ and CanL− groups (Supplementary Fig. S1A, S1B and Table S1). Cut-off values for rK39 and SPLA were 0·127 and 0·074, respectively. These cut-offs enabled a Se of 100% (29/29) for both antigens with Sp of 100% in both cases (0/121) (Supplementary Table S1). When the cut-offs were applied to the European cohort, they originated a global seropositivity of 43·8% (168/384) and 2·9% (11/384) for the individual antigens, SPLA and rK39, respectively (Table 1, Fig. 2A–C). If seropositivity to both antigens was used as a requisite, then 2·3% (9/384) of the cohort was seropositive (Table 1).

Fig. 2. Reactivity of CanL+, CanL− and European dog sera against SPLA and rK39 antigens (A). Correlation of the seroreactivity against SPLA and rK39 antigens in CanL+ and CanL− cohorts (B) and in European cohort (C). Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n). Bars represent the median for the cohort and each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate.

Table 1. Seropositivity levels in the different study cohorts for SPLA and rK39 antigens

a Positive for both antigens simultaneously.

b Positive for at least one of the antigens.

Observing the generated seropositivity data disparity for antigens that excelled at discriminating CanL from non-CanL dogs, two possibilities were considered: either the SPLA was detecting FP or rK39 was detecting FN. Considering that both antigens presented very high Sp and Se, the cut-off stringency was increased to decrease the number of FP (Supplementary Table S2 and Fig. 3A). For seropositivity associated with both antigens to have epidemiological relevance, then the increase in cut-off stringency should enable a decrease of FP associated with SPLA leading seropositivity levels similar to rK39. In the European cohort, using two cut-offs (cut-off value × 2) for rK39, the number of seropositive animals is reduced to just one, PT30 (Fig. 3B). For SPLA, 66/384 (17·2%) animals remained seropositive when two cut-offs were used (Supplementary Table S2). Comparing these SPLA-seropositive animals with the original 11 animals seropositive for rK39, only six animals were seropositive to rK39 and SPLA using two SPLA cut-offs (Fig. 3B). From 3 to 7 SPLA cut-offs, only three animals were seropositive for both antigens – when compared once again to the original rK39-positive animals (Fig. 3B). The sample with highest seropositivity to both antigens was PT30 (Fig. 3B).

Fig. 3. Representation of seroreactivity against SPLA and rK39 antigens in CanL+ and CanL+ cohorts (A), and European cohort (B). Vertical and horizontal dotted lines represent the number of cut-offs (n × cut-off) for each antigen. The cut-off-dependent sensitivity is represented in (A) for SPLA and for rK39. Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n). Each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate.

The geographic distribution of seropositive animals was not random, ranged from 0 to 86%. Poland presented (43/50) seropositive animals, while France presented 0/50 animals to SPLA (Fig. 4 and Supplementary Table S2). Considering the individual countries, rK39-seropositive animals were mostly from Portugal: 66·3% (7/11) (Fig. 4A, Supplementary Fig. S2A). Still, three animals were from non-endemic countries [two from Denmark (DK44 and 50) and one from Poland – PL9] and a final one from France (FR2) (Fig. 3). For SPLA, although there was also an over-representation of dogs from Portugal (i.e. 12·5%; 21/168), this was not as evident as for rK39 (Supplementary Fig. S2). As the groups were proportional, the basal cohort serological response was compared to evaluate if existed a regional influence in the response to the antigens (Supplementary Tables S3 and S4). For rK39, the dogs from Germany had a basal median response significantly different from Hungary and Portugal, while the animals from Portugal were significantly different from all the other cohorts (Supplementary Fig. S2A and Table S3). For SPLA, more countries had significant differences, with the cohort from Poland being the most divergent (Supplementary Fig. S2 and Table S4). As SPLA is an undefined antigen mixture, these variations in seropositivity might correspond to an unspecific response.

Fig. 4. Reactivity of dog sera against SECA antigen for: (A) individual countries Hungary (HU), The Netherlands (NL), UK, Germany (DE), France (FR), Denmark (DK) and Poland (PL). Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n) and (B) CanL+, CanL− and European cohort. Bars represent the median for the cohort and each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate. Statistical analysis was done by one-way ANOVA and Kruskal–Wallis tests. Statistical significance between groups is ‘extremely significant’ (***0·0001 ⩽ P < 0·001).

To address serological response Sp, an undefined ubiquitous non-related antigen was used, E. coli DH5-α, to evaluate if the serological response pattern was similar to SPLA suggesting a general unspecific response (Fig. 4A and Supplementary Fig. S2B). The SECA antigen had some predictive value to distinguish the control cohorts (Se of 81·5% and Sp of 71·9%) (Fig. 4B and Supplementary Table S1). Notwithstanding, the seropositivity profile against SECA was distinct from the SPLA (Fig. 4A, Supplementary Fig. S2B and Tables S4 and S5). To further address the relationship between these antigens, correlations in the control cohorts and also in each individual country were done (Supplementary Table S6). In the CanL+ cohort, the highest correlation was obtained for rK39 vs SPLA (r = 0·499, P < 0·01; r 2 = 0·249), whereas the lowest was for rK39 vs SECA (r = −0·010, P = 0·952, r 2 = 0·0001). In the CanL− cohort, the highest correlation value was for SPLA vs SECA (r = 0·356, P < 0·001, r 2 = 0·127). At the country level, the most common significant correlation was SPLA vs SECA (3/8), while rK39 correlated positively with SPLA in two countries and with SECA in one country (Supplementary Table S6). The cohort comparison at a country level (absolute values and correlation data) suggests distinct basal responses to the different antigens influencing the serology absolute values.

To bypass the regional effects on the seropositivity, the ratios between the antigens were analysed for their capacity to predict CanL in the control cohorts. For this, the median normalized single-antigen reactivity in the CanL+ control group was compared (Figs 2 and 4B). The nrK39 and nSPLA responses were higher than nSECA in CanL+ cohort. Moreover, this was characteristic of the individual sera from the CanL+ cohort: (27/29) for nSPLA > nSECA and (26/29) nrK39 > nSECA (Supplementary Table S7). Likewise, the response to rK39 was higher than to SPLA in (24/29) of the cohort (Supplementary Table S7). Therefore, these ratios, [log(nSPLA/nSECA) > 1; log(nrK39/nSECA) > 1; log(nrK39/nSPLA) > 1] were characteristic of CanL dogs (Supplementary Fig. S3). Consequently, ROC curves analysis was performed for each of the aforementioned individual ratios (Supplementary Fig. S1D–F). The predictive value of these ratios was considerable, although not superior overall to the reference antigens alone (Supplementary Table S1). The Sp of the ratios was 94·2, 92·6 and 72·7%, for nSPLA/nSECA, nrK39/nSECA and nrK39/nSPLA, respectively (Supplementary Tables S1 and S8). The lower Se of the assays can be attributed to artefactual responses due to low serological responders that create ratios that are above the cut-off level (see PL38, FR9 and DE47 in Supplementary Table S9). Therefore, the capacity of these ratios to predict disease requires the determination of positivity to reference antigens to exclude the aforementioned artefactual values. Taking this in consideration, a scoring system was created to address not only the positivity to the reference antigens but also to the characteristic ratios described above (Fig. 1). The Cohen's κ coefficient was determined for the possible scores to define the best predictive value when compared to both reference antigens. The scores of 4 or 3 presented the best predictive value (Supplementary Table S10). We excluded the score 3 as it enabled positive results that were negative for both reference antigens (Fig. 1). This approach (using a score ⩾4) resulted in an overall Se (96%) and Sp (100%) similar to the combined reference antigens (Table 2). When this score system was applied to our cohorts (Table 2), it reduced the number of seropositive animals in the European cohort to seven (PT10, PT30, PL9, PL24, DK43, DK50 and FR2) with an overall seropositivity of 1·8% (7/384) (Table 2 and Supplementary Tables S9–12). This represents a diminution of 36% in seropositivity when compared with rK39 alone. We also report a diminution of 22% in seropositivity when compared with rK39 and SPLA combined (Table 1). Only one animal in Europe presented a perfect score of 5 (PT30), the most common score in the CanL+ cohort [82·8% (24/29)], with the remaining six animals presenting a score of 4. These seven seropositive animals from the European cohort obtained after application of the score methodology were all rK39-seropositive animals with the exception of one sample from Poland (PL24) (Supplementary Fig. S4 and Table S9).

Table 2. Seropositivity levels in the different study cohorts using the scoring system

a 5: seropositivity to all serological parameters defined in Fig. 1.

b 4: seropositivity to four out of five serological of the parameters defined in Fig. 1.

c Total: seropositivity to at least four out of the parameters defined in Fig. 1.

DISCUSSION

In the present study, the used antigens, SPLA and rK39, are established antigens with reported high sensitivities and specificities and are the base of several reference tests. The cut-off values for two antigens, using a cohort of CanL+ dogs and a cohort of CanL− dogs, were determined. The obtained cut-offs were similar in absolute value to the ones obtained in previous work from our group (Santarem et al. Reference Santarem, Silvestre, Cardoso, Schallig, Reed and Cordeiro-da-Silva2010), in a clear indication that both antigens are capable of consistently discriminating CanL+ from CanL− dogs. When the newly determined cut-offs were applied to a cohort of animals from Europe without clinical characterization, a seropositivity that was distinct for both antigens (43·8% for SPLA and 2·9% for rK39) was obtained. This large difference in seropositivity cannot be attributed to laboratory inter variability, as all assays were performed in the same laboratory, including the ROC curve analysis. The CanL seropositivity levels reported for endemic Mediterranean regions range between 5 and 30% (Moreno and Alvar, Reference Moreno and Alvar2002). The overall seropositivity levels obtained for the non-endemic countries was not in accordance with epidemiological data that do not support significant seropositivity levels in the non-endemic countries from the European cohort (Solano-Gallego et al. Reference Solano-Gallego, Miro, Koutinas, Cardoso, Pennisi, Ferrer, Bourdeau, Oliva and Baneth2011). The rK39 seropositivity levels are more in the range of what is reported from the endemic regions, but still non-endemic seropositive samples were identified in Denmark and Poland. These positive animals might be associated with canine population mobility associated with travelling, an issue of growing concern for CanL (Menn et al. Reference Menn, Lorentz and Naucke2010; Bart et al. Reference Bart, van Thiel, de Vries, Hodiamont and Van Gool2013). L eishmania infantum is endemic in southern European countries (Dujardin et al. Reference Dujardin, Campino, Canavate, Dedet, Gradoni, Soteriadou, Mazeris, Ozbel and Boelaert2008) that are a highly valued vacation destination generally during the peak of the transmission season. In these conditions, the risk for exposure to the parasite can be as high as 0·23% for dogs coming from northern Europe (Teske et al. Reference Teske, van Knapen, Beijer and Slappendel2002). In fact, the few reported CanL cases in northern Europe are restricted to dogs that have travelled to endemic areas of the Mediterranean basin during periods when phlebotomine sand fly exposure is high, mostly between March and November, or that have to be relocated from those areas (Saari et al. Reference Saari, Rasi and Anttila2000).

SPLA in known to have cross-reactivity with other diseases like babesiosis, Chagas, ehrlichiosis, rickettsiosis and toxoplasmosis, probably due to some epitope conservation generating unspecificity (Gomes et al. Reference Gomes, Paiva Cavalcanti, Lira, Abath and Alves2008; Saridomichelakis, Reference Saridomichelakis2009; Silvestre et al. Reference Silvestre, Santarem, Teixeira, Cunha, Schallig and Cordeiro-da-Silva2009; Zanette et al. Reference Zanette, Lima, Laurenti, Rossi, Vides, Vieira, Biondo and Marcondes2014; Peixoto et al. Reference Peixoto, de Oliveira and Romero2015). This cross-reactivity was suggested upon correlation of the data from SPLA with SECA (Supplementary Table S6). In the cohorts from three countries, there was a significant correlation between SECA and SPLA, more than between SPLA and rK39. This suggests that, in the absence of a Leishmania-specific serological response, the SPLA might also be recognized by other non-related infectious diseases or immune disorders. This is also suggested by the correlation existing in the CanL− control group for these two antigens. Still, the distinct pattern of basal recognition between SPLA and SECA (Fig. 4A and Supplementary Fig. S2B) is not suggestive of general unspecific recognition due to immunological disorders, like polyclonal activation. Intriguingly, it will also be important to address if the apparent lack of Sp of SPLA might also be true for other assays that use parasite total antigens, such as IFAT or DAT. The rK39 was less prone to cross-reactivity (Fig. 3B). In fact, the general basal serological response was more uniform. This is in agreement with the recognized value of rK39 as a good serological marker for leishmaniosis (Porrozzi et al. Reference Porrozzi, Santos da Costa, Teva, Falqueto, Ferreira, dos Santos, Fernandes, Gazzinelli, Campos-Neto and Grimaldi2007). Still, regional differences in antigen performance have been already reported, the performance of rK39 (for the human visceral leishmaniasis) is known to be diminished in sub-Saharan Africa (Pattabhi et al. Reference Pattabhi, Whittle, Mohamath, El-Safi, Moulton, Guderian, Colombara, Abdoon, Mukhtar, Mondal, Esfandiari, Kumar, Chun, Reed and Bhatia2010; Bezuneh et al. Reference Bezuneh, Mukhtar, Abdoun, Teferi, Takele, Diro, Jemaneh, Shiferaw, Wondimu, Bhatia, Howard, Ghalib, Ireton, Hailu and Reed2014; Mukhtar et al. Reference Mukhtar, Abdoun, Ahmed, Ghalib, Reed, Boelaert, Menten, Khair and Howard2015). Subtle genetic differences in Leishmania strains are sufficient to influence the performance of the recombinant antigens (Abass et al. Reference Abass, Kang, Martinkovic, Semiao-Santos, Sundar, Walden, Piarroux, El Harith, Lohoff and Steinhoff2015).

The adjustment of the cut-off stringency is often used to help in the diagnosis of CanL (Paltrinieri et al. Reference Paltrinieri, Solano-Gallego, Fondati, Lubas, Gradoni, Castagnaro, Crotti, Maroli, Oliva, Roura, Zatelli and Zini2010). Therefore, we increased the cut-off stringency (at the cost of decreased Se) (Fig. 3A). Two cut-offs decreased the global seropositivity for SPLA and rK39 to 66/384 (17·2%) and 1/384 (0·26%), respectively. Still, a significant difference in readout between both antigens was apparent, as there was no correspondence between SPLA- and rK39-seropositive animals. In fact, the increase in SPLA cut-off stringency also led to a loss of rK39-positive animals (standard cut-off), instead of having an enrichment of double positive sera as would be expected for seropositivity related to CanL. Still, it must be referred that existence of dogs with different clinical stage of the disease might also contribute to distinct levels of seropositivity to rK39 and SPLA. It was shown for naturally infected dogs that rK39, although adequate for detecting symptomatic infections, presented lower capacity to evaluate sub-clinical infections (Santarem et al. Reference Santarem, Silvestre, Cardoso, Schallig, Reed and Cordeiro-da-Silva2010). This could justify the failure in enrichment for double positive animals with the cut-off increase. Still, as most countries in the study are non-endemic (exception of Portugal and France), natural exposure to the parasite and asymptomatic infections should not be a decisive factor for the lack of correspondence in seropositivity levels.

To address the possibility of unspecificity of SPLA in the European cohort, SECA was used as a measure of the humoural response that is not specifically directed to Leishmania. Although SECA has some predictive value (probably as a consequence of some level of conservation at the epitope level or due to, in severe cases of the disease, polyclonal activation and effects of co-infections), it presented low Sp (71·9%). Upon cut-off normalization, it was also evident the clear relationships between the antigens. The median normalized response of the animals of the CanL+ control group was highest for rK39 and lowest for SECA (Figs 2A and 4B). This characteristic seropositivity pattern rK39 > SPLA > SECA was also evident for the individual animals (Supplementary Table S8). This was suggestive that the relation between the antigens might have predictive value for the detection of CanL. If fact, the relationship between the three antigens (nSPLA/nSECA, nrK39/nSECA, nrK39/nSPLA) presented significant predictive power, as determined be ROC curve analysis (Supplementary Table S1). Still, their direct application in the European cohort did not result in a diminution of seropositivity (Supplementary Table S8). This was not unexpected because the relation is independent on absolute values. Therefore, very low serological results might be seropositive using this approach. Therefore, the unrestricted use of antigen seropositivity ratios present a significant risk of irrelevant artefactual values. Still, these data clearly established that the ratios between the antigens followed a pattern that is in conjunction with positivity to one or both reference antigens was found to be predictive of CanL+ (Fig. 1).

When applied to the European cohort, the score approach originated a global seropositivity of 1·8% (7/384). These seven animals were indistinguishable from the CanL+ cohort in at least four evaluated characteristics (one presented a perfect score of 5). This was the animal sorted upon the increase of rK39 threshold (PT30). Only one animal from Poland (PL24), which was classified with a score of 4, was not positive for rK39 alone. This sample was positive for SPLA and for the three ratios evaluated in the score system (nSPLA/nSECA, nrK39/nSECA and nrK39/nSPLA). Although the absolute value of the OD to rK39 was below the cut-off (0·127) for this sample, this difference was only 0·021 (see PL24 in Supplementary Table S9). This is a clear example of problems associated with using absolute values. Subtle differences can place a serum in opposite sides of the threshold. For example, PT45 was seropositive for rK39, having 0·134 of average OD. Still, in our score system, it was negative with only a score of 3 due to a disproportionally high response to SECA and SPLA (Supplementary Table S9). The high reactivity to SECA prevented positive ratios and consequently preventing the higher score. This very high relative reactivity to SECA was not characteristic of our CanL+ cohort. PT34 is another interesting sample with overall high responses to all tested antigens with average ODs of 0·659 for SECA, 0·447 SPLA and 0·147 for rK39. Once again, this profile is not characteristic of the infected cohort and might reflect auto-immunity or other complications or a different stage of disease.

In the CanL+ group also, some samples are mentionworthy. The CanL+ 4 is the sample that prevents a 100% Se for the score approach. This is the sample with the highest SECA average OD (2·455). This might be due to an already advanced stage of the disease resulting in the loss of the characteristic serological pattern of active disease due to other co-infections or even polyclonal activation.

The presented data demonstrate that the evaluation of seropositivity is a complex issue that is often oversimplified. We report that complex mixtures of antigens like SPLA are prone to cross-reactivity making them more unreliable for serosurveillance studies than rK39. Recombinant antigens like rK39 were less afflicted by regional-specific serological background and still might be more sensitive to subtle strain-specific changes in the native antigen. The use of ratios associated with serological responses to specific antigens can be used to create more accurate serological profiles that might contribute for identification of infected animals. Disease-specific serological footprints might be exploited to better understand the immune responses of clinically and sub-clinically infected and/or merely exposed animals, enabling early warning system for disease development and more accurate serosurveillance studies. Further studies with different recombinant antigens and experimentally infected animals and cohorts of animals with different clinical status will validate these observations and help to overcome the reported failure of complex antigens mixtures (SPLA) and recombinant proteins (rK39) in establishing meaningful seropositivity levels.

SUPPLEMENTARY MATERIAL

The supplementary material for this article can be found at https://doi.org/10.1017/S0031182017000713.

ACKNOWLEDGEMENTS

Our thanks to Dr Steven Reed, from Infectious Disease Research Institute (Seattle, Washington, USA) for providing the recombinant protein rK39.

FINANCIAL SUPPORT

This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and from the European Community's Seventh Framework Programme under grant agreement No. 603181 [Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin)].

‘This article is a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)’.

C.L. and N.S. were supported by BD SFRH/BD/89183/2012 and European Community's Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED), respectively.

ETHICAL AND REGULATORY GUIDELINES

This study observed Portuguese legislation for the protection of animals (Law no. 92/1995, from 12 September). According to the European Directive of 24 November 1986, article 2 d, non-experimental, agricultural or clinical veterinary were excluded. The Animal Ethics Committee of the Associate Laboratory IBMC-INEB approved the animal protocol used. Serum samples were collected during vaccination campaigns, and informed consent was obtained from all dog owners before sample collection.

References

REFERENCES

Abass, E., Kang, C., Martinkovic, F., Semiao-Santos, S. J., Sundar, S., Walden, P., Piarroux, R., El Harith, A., Lohoff, M. and Steinhoff, U. (2015). Heterogeneity of Leishmania donovani parasites complicates diagnosis of visceral leishmaniasis: comparison of different serological tests in three endemic regions. PLoS ONE 10(3), e0116408. doi: 10.1371/journal.pone.0116408.CrossRefGoogle ScholarPubMed
Arruda, M. M., Figueiredo, F. B., Marcelino, A. P., Barbosa, J. R., Werneck, G. L., Noronha, E. F. and Romero, G. A. (2016). Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection. Memórias do Instituto Oswaldo Cruz 111(3), 168173. doi: 10.1590/0074-02760150364.CrossRefGoogle ScholarPubMed
Bart, A., van Thiel, P. P., de Vries, H. J., Hodiamont, C. J. and Van Gool, T. (2013). Imported leishmaniasis in the Netherlands from 2005 to 2012: epidemiology, diagnostic techniques and sequence-based species typing from 195 patients. Eurosurveillance 18(30), 8.CrossRefGoogle ScholarPubMed
Bezuneh, A., Mukhtar, M., Abdoun, A., Teferi, T., Takele, Y., Diro, E., Jemaneh, A., Shiferaw, W., Wondimu, H., Bhatia, A., Howard, R. F., Ghalib, H., Ireton, G. C., Hailu, A. and Reed, S. G. (2014). Comparison of point-of-care tests for the rapid diagnosis of visceral leishmaniasis in East African patients. American Journal of Tropical Medicine and Hygiene 91(6), 11091115. doi: 10.4269/ajtmh.13-0759.CrossRefGoogle ScholarPubMed
Carvalho, B. M., Rangel, E. F., Ready, P. D. and Vale, M. M. (2015). Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change. PLoS ONE 10(11), e0143282. doi: 10.1371/journal.pone.0143282.CrossRefGoogle ScholarPubMed
Courtenay, O., Carson, C., Calvo-Bado, L., Garcez, L. M. and Quinnell, R. J. (2014). Heterogeneities in Leishmania infantum infection: using skin parasite burdens to identify highly infectious dogs. PLoS Neglected Tropical Diseases 8(1), e2583. doi: 10.1371/journal.pntd.0002583.CrossRefGoogle ScholarPubMed
Dujardin, J. C., Campino, L., Canavate, C., Dedet, J. P., Gradoni, L., Soteriadou, K., Mazeris, A., Ozbel, Y. and Boelaert, M. (2008). Spread of vector-borne diseases and neglect of leishmaniasis, Europe. Emerging Infectious Diseases 14(7), 10131018. doi: 10.3201/eid1407.071589.CrossRefGoogle Scholar
Elshafie, A. I., Mullazehi, M. and Ronnelid, J. (2016). General false positive ELISA reactions in visceral leishmaniasis. Implications for the use of enzyme immunoassay analyses in tropical Africa. Journal of Immunological Methods 431, 6671. doi: 10.1016/j.jim.2016.02.007.CrossRefGoogle ScholarPubMed
Fraga, D. B., Pacheco, L. V., Borja, L. S., Tuy, P. G., Bastos, L. A., Solca Mda, S., Amorim, L. D. and Veras, P. S. (2016). The rapid test based on Leishmania infantum chimeric rK28 protein improves the diagnosis of canine visceral leishmaniasis by reducing the detection of false-positive dogs. PLoS Neglected Tropical Diseases 10(1), e0004333. doi: 10.1371/journal.pntd.0004333.CrossRefGoogle Scholar
Geisweid, K., Weber, K., Sauter-Louis, C. and Hartmann, K. (2013). Evaluation of a conjunctival swab polymerase chain reaction for the detection of Leishmania infantum in dogs in a non-endemic area. Veterinary Journal 198(1), 187192. doi: 10.1016/j.tvjl.2013.07.025.CrossRefGoogle Scholar
Gomes, Y. M., Paiva Cavalcanti, M., Lira, R. A., Abath, F. G. and Alves, L. C. (2008). Diagnosis of canine visceral leishmaniasis: biotechnological advances. Veterinary Journal 175(1), 4552. doi: 10.1016/j.tvjl.2006.10.019.CrossRefGoogle ScholarPubMed
Maia, C. and Cardoso, L. (2015). Spread of Leishmania infantum in Europe with dog travelling. Veterinary Parasitology 213(1–2), 211. doi: 10.1016/j.vetpar.2015.05.003.CrossRefGoogle ScholarPubMed
Menn, B., Lorentz, S. and Naucke, T. J. (2010). Imported and travelling dogs as carriers of canine vector-borne pathogens in Germany. Parasites & Vectors 3, 34. doi: 10.1186/1756-3305-3-34.CrossRefGoogle ScholarPubMed
Michalsky, E. M., Rocha, M. F., da Rocha Lima, A. C., Franca-Silva, J. C., Pires, M. Q., Oliveira, F. S., Pacheco, R. S., dos Santos, S. L., Barata, R. A., Romanha, A. J., Fortes-Dias, C. L. and Dias, E. S. (2007). Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies. Veterinary Parasitology 147(1–2), 6776. doi: 10.1016/j.vetpar.2007.03.004.CrossRefGoogle ScholarPubMed
Millan, J., Ferroglio, E. and Solano-Gallego, L. (2014). Role of wildlife in the epidemiology of Leishmania infantum infection in Europe. Parasitology Research 113(6), 20052014. doi: 10.1007/s00436-014-3929-2.CrossRefGoogle Scholar
Miro, G., Checa, R., Montoya, A., Hernandez, L., Dado, D. and Galvez, R. (2012). Current situation of Leishmania infantum infection in shelter dogs in northern Spain. Parasites & Vectors 5, 60. doi: 10.1186/1756-3305-5-60.CrossRefGoogle ScholarPubMed
Molina, R., Amela, C., Nieto, J., San-Andres, M., Gonzalez, F., Castillo, J. A., Lucientes, J. and Alvar, J. (1994). Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus . Transactions of the Royal Society of Tropical Medicine and Hygiene 88(4), 491493.CrossRefGoogle ScholarPubMed
Morales-Yuste, M., Morillas-Marquez, F., Diaz-Saez, V., Baron-Lopez, S., Acedo-Sanchez, C. and Martin-Sanchez, J. (2012). Epidemiological implications of the use of various methods for the diagnosis of canine leishmaniasis in dogs with different characteristics and in differing prevalence scenarios. Parasitology Research 111(1), 155164. doi: 10.1007/s00436-011-2812-7.CrossRefGoogle Scholar
Moreno, J. and Alvar, J. (2002). Canine leishmaniasis: epidemiological risk and the experimental model. Trends in Parasitology 18(9), 399405. doi: S1471492202023474 [pii].CrossRefGoogle ScholarPubMed
Mukhtar, M., Abdoun, A., Ahmed, A. E., Ghalib, H., Reed, S. G., Boelaert, M., Menten, J., Khair, M. M. and Howard, R. F. (2015). Diagnostic accuracy of rK28-based immunochromatographic rapid diagnostic tests for visceral leishmaniasis: a prospective clinical cohort study in Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 109(9), 594600. doi: 10.1093/trstmh/trv060.CrossRefGoogle ScholarPubMed
Naucke, T. J., Menn, B., Massberg, D. and Lorentz, S. (2008). Sandflies and leishmaniasis in Germany. Parasitology Research 103(Suppl. 1), S6568. doi: 10.1007/s00436-008-1052-y.CrossRefGoogle ScholarPubMed
Noli, C. and Saridomichelakis, M. N. (2014). An update on the diagnosis and treatment of canine leishmaniosis caused by Leishmania infantum (syn. L. chagasi). Veterinary Journal 202(3), 425435. doi: 10.1016/j.tvjl.2014.09.002.CrossRefGoogle Scholar
Palatnik-de-Sousa, C. B. (2012). Vaccines for canine leishmaniasis. Frontiers in Immunology 3, 69. doi: 10.3389/fimmu.2012.00069.CrossRefGoogle ScholarPubMed
Paltrinieri, S., Solano-Gallego, L., Fondati, A., Lubas, G., Gradoni, L., Castagnaro, M., Crotti, A., Maroli, M., Oliva, G., Roura, X., Zatelli, A., Zini, E. and Canine Leishmaniasis Working Group, I. S. o. V. o. C. A. (2010). Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Journal of American Veterinary Medical Association 236(11), 11841191. doi: 10.2460/javma.236.11.1184.CrossRefGoogle ScholarPubMed
Parikh, R., Mathai, A., Parikh, S., Chandra Sekhar, G. and Thomas, R. (2008). Understanding and using sensitivity, specificity and predictive values. Indian Journal of Ophthalmology 56(1), 4550.CrossRefGoogle ScholarPubMed
Pattabhi, S., Whittle, J., Mohamath, R., El-Safi, S., Moulton, G. G., Guderian, J. A., Colombara, D., Abdoon, A. O., Mukhtar, M. M., Mondal, D., Esfandiari, J., Kumar, S., Chun, P., Reed, S. G. and Bhatia, A. (2010). Design, development and evaluation of rK28-based point-of-care tests for improving rapid diagnosis of visceral leishmaniasis. PLoS Neglected Tropical Diseases 4(9), e822. doi: 10.1371/journal.pntd.0000822.CrossRefGoogle ScholarPubMed
Peixoto, H. M., de Oliveira, M. R. and Romero, G. A. (2015). Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis. Tropical Medicine & International Health 20(3), 334352. doi: 10.1111/tmi.12429.CrossRefGoogle ScholarPubMed
Porrozzi, R., Santos da Costa, M. V., Teva, A., Falqueto, A., Ferreira, A. L., dos Santos, C. D., Fernandes, A. P., Gazzinelli, R. T., Campos-Neto, A. and Grimaldi, G. Jr. (2007). Comparative evaluation of enzyme-linked immunosorbent assays based on crude and recombinant leishmanial antigens for serodiagnosis of symptomatic and asymptomatic Leishmania infantum visceral infections in dogs. Clinical and Vaccine Immunology 14(5), 544548. doi: 10.1128/CVI.00420-06.CrossRefGoogle ScholarPubMed
Ready, P. D. (2010). Leishmaniasis emergence in Europe. Eurosurveillance 15(10), 11.Google Scholar
Saari, S., Rasi, J. and Anttila, M. (2000). Leishmaniosis mimicking oral neoplasm in a dog: an unusual manifestation of an unusual disease in Finland. Acta Veterinaria Scandinavica 41(1), 101104.Google Scholar
Santarem, N., Silvestre, R., Cardoso, L., Schallig, H., Reed, S. G. and Cordeiro-da-Silva, A. (2010). Application of an improved enzyme-linked immunosorbent assay method for serological diagnosis of canine leishmaniasis. Journal of Clinical Microbiology 48(5), 18661874. doi: 10.1128/JCM.02402-09.CrossRefGoogle ScholarPubMed
Saridomichelakis, M. N. (2009). Advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications. Veterinary Dermatology 20(5–6), 471489. doi: VDE823 [pii], 10.1111/j.1365-3164.2009.00823.x.CrossRefGoogle ScholarPubMed
Silva, D. T. d., Starke-Buzetti, W. A., Alves-Martin, M. F., Paixão, M. d. S., Tenório, M. d. S. and Lopes, M. L. M. (2014). Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis. Revista Brasileira de Parasitologia Veterinária 23(2), 179186. doi: 10.1590/s1984-29612014033.CrossRefGoogle ScholarPubMed
Silvestre, R., Santarem, N., Teixeira, L., Cunha, J., Schallig, H. and Cordeiro-da-Silva, A. (2009). Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis. American Journal of Tropical Medicine and Hygiene 81(2), 202208. doi: 81/2/202 [pii].Google ScholarPubMed
Solano-Gallego, L., Miro, G., Koutinas, A., Cardoso, L., Pennisi, M. G., Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G. and The LeishVet, G. (2011). LeishVet guidelines for the practical management of canine leishmaniosis. Parasites & Vectors 4, 86. doi: 10.1186/1756-3305-4-86.CrossRefGoogle ScholarPubMed
Sousa, S., Lopes, A. P., Cardoso, L., Silvestre, R., Schallig, H., Reed, S. G. and Cordeiro da Silva, A. (2011). Seroepidemiological survey of Leishmania infantum infection in dogs from northeastern Portugal. Acta Tropica 120(1–2), 8287. doi: 10.1016/j.actatropica.2011.06.003.CrossRefGoogle ScholarPubMed
Teske, E., van Knapen, F., Beijer, E. G. and Slappendel, R. J. (2002). Risk of infection with Leishmania spp. in the canine population in the Netherlands. Acta Veterinaria Scandinavica 43(4), 195201.CrossRefGoogle ScholarPubMed
Wylie, C. E., Carbonell-Antonanzas, M., Aiassa, E., Dhollander, S., Zagmutt, F. J., Brodbelt, D. C. and Solano-Gallego, L. (2014). A systematic review of the efficacy of prophylactic control measures for naturally-occurring canine leishmaniosis, part I: vaccinations. Preventive Veterinary Medicine 117(1), 718. doi: 10.1016/j.prevetmed.2014.06.015.CrossRefGoogle ScholarPubMed
Zanette, M. F., Lima, V. M., Laurenti, M. D., Rossi, C. N., Vides, J. P., Vieira, R. F., Biondo, A. W. and Marcondes, M. (2014). Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs. Revista da Sociedade Brasileira de Medicina Tropical 47(1), 105107. doi: S0037-86822014000100105 [pii], 10.1590/0037-8682-1723-2013.CrossRefGoogle ScholarPubMed
Figure 0

Fig. 1. Evaluation of seropositivity of each individual serum evaluated as a cumulative score. Each individual serum was ‘scored’ as nSPLA + nrK39 + nSECA + SPLA/SECA + rK39/SECA + rK39/SPLA using the following premise: each serology value above or equal to the cut-off values of nSPLA, nrK39, nSECA, SPLA/SECA, rK39/SECA and rK39/SPLA was transformed into a score of ‘1’ originating a cumulative score ranging from 0 to 5 to each individual sample.

Figure 1

Fig. 2. Reactivity of CanL+, CanL− and European dog sera against SPLA and rK39 antigens (A). Correlation of the seroreactivity against SPLA and rK39 antigens in CanL+ and CanL− cohorts (B) and in European cohort (C). Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n). Bars represent the median for the cohort and each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate.

Figure 2

Table 1. Seropositivity levels in the different study cohorts for SPLA and rK39 antigens

Figure 3

Fig. 3. Representation of seroreactivity against SPLA and rK39 antigens in CanL+ and CanL+ cohorts (A), and European cohort (B). Vertical and horizontal dotted lines represent the number of cut-offs (n × cut-off) for each antigen. The cut-off-dependent sensitivity is represented in (A) for SPLA and for rK39. Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n). Each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate.

Figure 4

Fig. 4. Reactivity of dog sera against SECA antigen for: (A) individual countries Hungary (HU), The Netherlands (NL), UK, Germany (DE), France (FR), Denmark (DK) and Poland (PL). Results are expressed as the logarithm of the optical density (OD) at 492 nm normalized by the cut-off value for each antigen (n) and (B) CanL+, CanL− and European cohort. Bars represent the median for the cohort and each single dot represents the dataset for an individual dog. The data depicted were generated from the average of at least two independent assays done in triplicate. Statistical analysis was done by one-way ANOVA and Kruskal–Wallis tests. Statistical significance between groups is ‘extremely significant’ (***0·0001 ⩽ P < 0·001).

Figure 5

Table 2. Seropositivity levels in the different study cohorts using the scoring system

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