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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2019 Oct 23;57(11):e00819-19. doi: 10.1128/JCM.00819-19

Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs

A E Bivona a,b,#, L Czentner a,b,#, A Sanchez Alberti a,b, N Cerny b, A C Cardoso Landaburu a,b, C Nevot c, O Estévez c, J D Marco d, M A Basombrio d, E L Malchiodi a,b, S I Cazorla b,e,✉
Editor: Michael J Loeffelholzf
PMCID: PMC6813014  PMID: 31434727

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions.

Keywords: Leishmania braziliensis, cysteine proteinase B, canine leishmaniasis serodiagnosis

ABSTRACT

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

INTRODUCTION

Leishmaniasis is endemic in 88 countries, with an estimated 350 million people at risk of becoming infected. Leishmaniasis is transmitted by the bite of infected female phlebotomine sandflies and is caused by different flagellate protozoans of the family Trypanosomatidae belonging to the genus Leishmania (1). These intracellular protozoa have a complex digenetic life cycle, requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The main epidemiological reservoirs of Leishmania infantum are dogs, which can remain asymptomatic for long periods of time, to finally develop cutaneous or systemic symptoms (2, 3). In Latin America, canine leishmaniasis is widespread, being one of the most important canine zoonotic vector-borne diseases (4).

More than 20 species and subspecies of Leishmania infect humans and dogs, causing a wide spectrum of diseases, including cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and visceral leishmaniasis (VL), depending on the parasite virulence factors and the immune response established by the host (5). In America, CL, MCL, and DCL taken together are also known as American tegumentary leishmaniasis (TL), with a wide geographical distribution from the southern United States to northern Argentina. In Northwest Argentina (NWA), there have been several CL outbreaks, mainly in the forest of Salta (6, 7).

In 2006, the first autochthonous human VL case was reported in Posadas, province of Misiones (northeastern Argentina [NEA]) (8, 9). Since then, climate change has contributed to the spread of VL in Argentina. Dogs have been found to be naturally infected with species such as Leishmania (Viannia) peruviana, Leishmania (Leishmania) major, and Leishmania (Leishmania) tropica, among others, in several countries (10). In Argentina, Leishmania (Viannia) braziliensis and Leishmania infantum have been incriminated as the causal agents of canine leishmaniasis in the cities of Orán and Posadas, NWA and NEA, respectively (11–13).

Traditionally, the diagnosis of leishmaniasis is based on the microscopic detection of amastigotes in tissue macrophages obtained by aspiration, scraping, or skin biopsy for CL and in bone marrow, nodes, and spleen for VL. However, the presence of amastigotes depends on several factors, and they can be morphologically misidentified as fungi, Toxoplasma, Histoplasma, or even artifacts (14). To increase diagnostic sensitivity and specificity, cultured lesion material and molecular biology techniques such as PCR and real-time quantitative PCR (qPCR) have been proposed (15, 16). However, not all Leishmania strains grow at the same rate, and not all tissues have similar parasite loads. Moreover, these techniques are expensive and require sophisticated laboratories.

As VL infection develops, large amounts of polyclonal antibodies are produced in the host (hypergammaglobulinemia). Therefore, various methods of detection of nonspecific antibodies have been used, which have subsequently been discarded for lack of sensitivity and specificity. Other methods such as electrophoresis, hemagglutination, the complement fixation test, and the gel diffusion test have been performed in different areas of endemicity. Currently, only the direct agglutination test, the immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatography are being used (17–19). Improving serological tests for the diagnosis of leishmaniasis is important because they are rapid, easy to perform, and can be easily implemented under the conditions commonly encountered in developing countries.

Antibodies against a wide range of parasitic antigens such as rK39 (a kinesin-related antigen), rK9, and rK26, heat shock proteins (HSP-70), histones (H-2A, 2B-H, H-3, and and H-4), cysteine proteinases (CPA and CPB), gp63 and gp70 proteins, ribosomal proteins P (P0, P2a, and P2b), iron superoxide dismutases (Fe-SODe), and the cathepsin L-like protein, among others, have been detected in Leishmania species infections (20–23). The rK39 antigen is one of the most used antigens for the diagnosis of canine and human VL, showing excellent results mainly in India, where sensitivity and specificity are almost 100% (24–26). Although antigen rK39 has been important for VL serodiagnosis, it does not allow the diagnosis of CL or MCL (27, 28).

The identification of new antigens to be employed in sensitive and specific serological assays is highly desirable. Extensive studies on the parasitic protozoan Leishmania have shown that cysteine proteinases (CPs) are involved in parasite survival, replication, and the onset of disease (29). The cysteine proteinase B (CPB) from Leishmania spp. is present in all strains and stages of the parasite and plays a crucial role in host-parasite interaction. The genes that code for the CPBs in trypanosomatids are organized as follows: a preregion, a propeptide, the catalytic domain, and a C-terminal extension (30, 31). The latter, as those of other CP orthologues, presents different immunogenic properties. We have demonstrated that the immune response in Trypanosoma cruzi infection is directed mostly against the C-terminal domain (32). This part of the antigen may operate as a diversion of the immune system, concentrating the antibody response against the C-terminal domain, and preserving the enzymatic activity of the N-terminal domain. Accordingly, our overall objective was to contribute to the diagnosis of cutaneous and visceral leishmaniasis in dogs using the recombinant CPB from L. braziliensis and its domains for the detection of specific antibodies against Leishmania spp.

MATERIALS AND METHODS

Cloning, expression, and purification of CPB and its domains in prokaryotic cells.

The cloning of the recombinant proteins will be described elsewhere (A. E. Bivona, unpublished results). Briefly, the CPB gene of L. braziliensis (LbrM08_V2.0820, accession XM_001562090) was synthesized by GenScript, optimizing the sequence between nucleotides 373 and 954 for expression in prokaryotic cells. From this gene, using specific primers containing cleavage sites for restriction enzymes and a tail of six histidines, we synthesized by PCR sequences of 954, 657, and 297 bp corresponding to the full-length CPB and its N-and C- terminal domains, respectively. The purified PCR products were digested with restriction enzymes and ligated to plasmid pET23a. Bacterium Escherichia coli DH5 was transformed with the constructs, and after selecting positive clones for their resistance to ampicillin, the presence of the inserts was confirmed by digestion with restriction enzymes. Constructs showed at least 97% identity with the previously reported sequence (LbrM08_V2.0820) for the entire CPB and N- and C-terminal domains.

The resulting vectors were then transformed into E. coli BL21(DE3) cells for expression. Recombinant proteins were obtained by inducing bacterial cultures with 1 mM isopropyl-l-thio-β-d-galactoside (IPTG) for 4 h. Cells were harvested, centrifuged, and resuspended in lysis buffer (pH 8.0) containing 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 μM E-64. The cells were stirred at room temperature for 60 min and then centrifuged at 10,000 × g for 20 min to pellet the cell debris. Proteins were purified under denaturing conditions from the supernatant using a Ni2+-nitrilotriacetic acid-Sepharose matrix. Properly folded proteins were obtained by extensive dialysis against buffer (2 M urea, 50 mM Tris, 5% sucrose, 10% glycerol, 0.3 M NaCl, 0.5 mM EDTA) followed by dialysis in phosphate-buffered saline (PBS)-20% glycerol and stored at −70°C until use. Protein concentration was determined by the Bradford protein assay (Bio-Rad, Hercules, CA), using bovine serum albumin (Sigma) as a standard.

Dog serum samples.

(i) Study 1. Samples were taken in the localities of Colonia Santa Rosa, Pichanal, and Orán, Province of Salta, NWA. The study area is included within the biogeographic Yungas rainforest (6). The Province of Salta has been the area of Argentina with high incidence of CL, with most cases originating in the Orán Department (7, 33, 34). Moreover, L. braziliensis has been acknowledged as the main causative agent for CL in this area of Argentina (7, 33).

Samples stored at −20°C, were collected from 76 dogs previously diagnosed with leishmaniasis by the identification of amastigotes in Giemsa-stained material obtained by touch print, scraping, exudate, or aspirate obtained by injecting 0.1 to 0.4 ml of buffered saline solution plus penicillin-streptomycin followed by aspiration of the fluid (6). Clinical signs supported the diagnosis of CL (6, 35). The entire skin surfaces of the dogs were carefully inspected for lesions or scars. Particular attention was paid to the limbs, ears, nose, and scrotum, since ulcerous lesions were most often found in these areas. The clinical criteria used to define “suspected leishmaniasis lesions” were ulcerative character, long duration, and rounded, raised, and indurated edges, coupled with swollen lymph nodes. Lesions probably induced by trauma were not considered to be Leishmania species infection.

Canines classified as no leishmaniasis (noL) were dogs without any sign of leishmaniasis and negative for the ELISA serological test (6).

(ii) Study 2. Thirty-three dogs were submitted to a careful clinical evaluation by veterinarians from Veterinaria del Oeste in the city of Posadas, Province of Misiones, NEA, and diagnosed with VL or no leishmaniasis (NoL) based on parasitological and serological tests and supported by clinical signs (36). We recently found Leishmania (Leishmania) infantum as the causative agent of canine VL cases in the city of Posadas (37).

Amastigotes observed on smears from aspirates were analyzed for the parasitological diagnosis of canine VL. A puncture aspiration was aseptically performed on the dogs using 2.5-ml syringes and 21-gauge needles. The aspirates were taken from enlarged lymph nodes, especially the popliteal ones. When lymph nodes could not be found, the samples were taken from the bone marrow or the spleen. A fraction from each sample obtained by aspiration or the scrapings was stained with Giemsa and observed under an optical microscope.

Clinical suspicion of VL was defined by the presence of three or more of the following signs: weight loss, alopecia, lymphadenopathy, renal azotemia, onychogryphosis, hepatomegaly, and splenomegaly. Signs such as exfoliative dermatitis on the nose, tail, and ear tips were also recorded. Skin features such as periocular and generalized alopecia, hair loss, seborrhea, and depigmentation in the muzzle were recorded to note the presence of skin disease without ulceration. Asymptomatic dogs appeared completely healthy at the clinical examination (no blood counts performed).

Blood samples were collected from the jugular vein and sera were kept frozen until tested. The diagnosis of canine VL was confirmed in the laboratory based on the positive results of IFAT, antigen rk39 (Kalazar Detect rapid test, canine; InBios International, Inc.) and SNAP Leishmania (IDEXX) (38).

Additionally, sera from dogs living in an area where VL is not endemic (Buenos Aires Province) not presenting any clinical signs of leishmaniasis, and negative by the serological evaluation were also included.

Ethics statement.

The dog owners voluntarily requested the medical attention of their animals. Under clinical suspicion of the disease, they gave their informed consent to include the dogs in this study. The procedures were approved by the Bioethics Committee of the Faculty of Agricultural and Veterinary Sciences, the Catholic University of Salta, Argentina (number 442837/0052, 14 October 2014).

Parasites.

Leishmania braziliensis promastigotes (MHOM/BR/75/M2903 strain) were grown in liver infusion tryptose (LIT) medium, which was prepared as follows: 5 g/liter liver infusion (2023-072K1066; Sigma), 5 g/liter tryptose (Britania), 2 g/liter glucose (Sigma), 68 mM NaCl, 5.4 mM KCl, 22 mM HPO4Na2, supplemented with 20 mg/liter hemin (Sigma) and 10% (vol/vol) fetal calf serum (FCS) (Internegocios). Culture maintenance was performed by weekly passages at 26°C.

Leishmania braziliensis supernatant lysate.

Promastigotes of L. braziliensis were centrifuged for 15 min at 5,000 × g, resuspended in 0.25 M sucrose, 5 mM KCl containing protease inhibitors (2 μM PMSF, 5 μM leupeptin, 5 μM pepstatin, and 5 μM E-64; Sigma, St. Louis, MO), and broken by three cycles of freeze-thawing and sonicating (4 cycles of 30 s on ice). The homogenate obtained was centrifuged at 45,000 × g, obtaining a supernatant fraction called F45 that was kept at −20°C until use. Protein concentration determination was performed by the Bradford method (protein assay, catalog number 500-0006; Bio-Rad), using bovine serum albumin as a standard.

IFAT.

Leishmania braziliensis promastigotes harvested during the exponential growth phase by centrifugation at 5,000 × g for 15 min were washed three times with 0.1 M phosphate-buffered saline (PBS; pH 7.2) and resuspended with 2% formalin solution in PBS. Formalin-treated promastigotes (1 × 105 parasites/field) placed in immunofluorescence glasses were fixed by heat, washed twice with PBS and finally with H2O. Sera were assayed at a 1/60 dilution, added to the coverslips, and incubated for 16 h at 4°C. As secondary antibody, anti-dog IgG (whole molecule)-fluorescein isothiocyanate (FITC) antibody produced in rabbit (F4012; Sigma) in 0.001% Evans blue was used and observed under a fluorescence microscope.

ELISA.

An indirect ELISA for antibody detection was used as described elsewhere (39). Briefly, flat polystyrene-bottom plates (Nunc, Roskilde, Denmark) were sensitized with 1 μg per well of the soluble fraction of L. braziliensis (F45) promastigote lysate or with 0.2 μg per well of the full-length CPB (F-CPB) and its N and C-terminal domains (N-CPB and C-CPB). Blocking was performed with 3% bovine serum albumin (BSA) and 0.1% gelatin in PBS for 1 h at 37°C. Plates were then washed three times with 0.05% Tween in PBS. Sera were assayed at a serial dilution of 1/100 and incubated for 18 h at 4°C. Peroxidase-conjugated immunoglobulins to dog IgG (Sigma) diluted 1/25,000 were used as a secondary antibody. Plates were developed by adding o-phenylenediamine (OPD)-H2O2 and incubating them for 10 min in the dark; the reaction was stopped using 4 N H2SO4. Optical density was read by an ELISA reader (Bio-Rad Laboratories, Hercules, CA) at 490 nm. Cutoff values were calculated using receiver operating characteristic (ROC) curves. Titers were calculated as the dilution in which the optical density (OD) obtained was equal to the mean of the controls ± 2.23 standard deviations (SDs; equivalent to 99% confidence in the one-tailed test hypothesis), where applicable.

Multiple sequence alignment.

The amino acid sequence of the L. braziliensis CPB (XP_001562140.1), without the prodomain region (amino acids [aa] 1 to 124), was aligned with the sequences registered in the NCBI database as “cpb” or “cysteine proteinase b” from other Leishmania species. Namely, L. guyanensis (ACS66748.1), L. panamensis (ABX74953.1), L. major (XP_001681135.1), L. infantum (SUZ39418.1), L. donovani (AGI92544.1), L. mexicana (CAA90236.1), L. tropica (AFN27127.1), and L. aethiopica (AAZ23596.1). Multiple sequence alignment was performed, and the phylogenetic tree was constructed using the ClustalW2 software tool (40).

Statistics.

The cutoff points for optimal sensitivity and specificity, as well as the other statistical parameters, were determined using the receiver operating characteristic (ROC) curve analysis to assess ELISA F-CPB, ELISA N-CPB, and ELISA C-CPB using the XL-STAT statistical software program (Excel).

Graphs were generated using the GraphPad Prism program (version 5.0). Statistical comparisons between groups were performed using the Mann-Whitney U test. P values of <0.05 were considered statistically significant.

RESULTS

CPB and its domains in the diagnosis of canine CL.

Dogs from northwestern Argentina were previously checked for lesions compatible with CL and for parasite microscopic observation of stained material from lesions (6). Accordingly, sera were classified as cutaneous leishmaniasis (CL) and no leishmaniasis (NoL) sera. We analyzed 76 stored serum samples by the immunofluorescence antibody test (IFAT). In slides containing fixed promastigotes of Leishmania braziliensis, a cutoff value of 1/60 was established for the in-house IFAT test. Later, we analyzed the samples, finding reactivity in 98.15% of the dogs diagnosed with CL. In contrast, 18.18% of dogs without leishmaniasis were positive against promastigotes of L. braziliensis by IFAT (Fig. 1). These results indicate values of sensitivity (Se) of 98.1% and specificity (Sp) of 81.8% for the IFAT test in the diagnosis of canine CL.

FIG 1.

FIG 1

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Immunofluorescence antibody test (IFAT) of dog sera from Northwest Argentina (NWA). Dogs previously diagnosed with cutaneous leishmaniasis (CL) (A) or no leishmaniasis (noL) (B) by direct methods, epidemiological, and clinical examination were tested for their reactivity against promastigotes of L. braziliensis by an IFAT. Fixed Leishmania braziliensis promastigotes were incubated overnight with dog sera with CL (C) and noL (D and E) and then stained with an anti-dog IgG FITC-labeled antibody. The figures show representative images of epifluorescence (C and D) and bright-field (E) microscopy. Magnification, ×40.

Titration curves were constructed to determine the most appropriate concentration of the L. braziliensis antigens to be used in the ELISAs (data not shown). Then, an ELISA was performed to determine specific IgG antibodies against the L. braziliensis promastigote lysate (F45), the recombinant full-length CPB (F-CPB), and its domains (N-CPB and C-CPB). Figure 2 shows that IgG-specific antibodies against F45, F-CPB, and its domains were significantly higher in CL than in nonleishmaniasis dogs (P < 0.0001).

FIG 2.

FIG 2

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ELISAs of sera from dogs living in Northwest Argentina. Canines classified as diagnosed with cutaneous leishmaniasis (CL) or no leishmaniasis (NoL) were assayed for the presence of IgG antibodies against L. braziliensis: (A) promastigote lysate (F45), (B) full-length CPB, (C) N-terminal domain of the CPB, and (D) C-terminal domain of the CPB. The results are expressed as the optical density at 490 nm (OD490), and the cutoff (CO) was calculated using the ROC curve. Lines represent the means ± standard errors of the means (SEMs). ****, P < 0.0001.

We analyzed the accuracy of the ELISAs to correctly classify the samples as CL. As shown in Fig. 3, areas under the curves (AUCs) of 0.9722 (95% confidence interval [CI], 0.9372 to 1.0070), 0.9722 (CI, 0.9347 to 1.010), 0.9562 (CI, 0.9055 to 1.007), and 0.9423 (CI, 0.8831 to 1.002) were determined for F45, F-CPB, N-CPB, and C-CPB, respectively. According to the traditional academic point system, all the antigens showed an AUC between 0.90 and 1.0, which means they were excellent ligands to correctly discriminate between the two groups (41).

FIG 3.

FIG 3

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ROC curves for ELISAs coated with the recombinant antigens. Sera from dogs from Northwest Argentina were analyzed by an ELISA against L. braziliensis: (A) promastigote lysate (F45), (B) full-length CPB, (C) N-terminal domain of the CPB, and (D) C-terminal domain of the CPB. True-positive rate (sensitivity) was plotted as a function of the false-positive rate (100% specificity) for the different Leishmania antigens at different cutoff points. An area of 1 represents a perfect test. while an area of 0.5 represents a worthless test. The accuracy of a diagnostic test is as follows: 0.90 to 1, excellent; 0.80 to 0.90, good; 0.70 to 0.80, fair; 0.60 to 0.70, poor; 0.50 to 0.60, fail.

Interestingly, the detection of antibodies against the recombinant antigens in the ELISA matrix showed sensitivities of 0.907, 0.944, and 0.943 for F-CPB, N-CPB, and C-CPB, respectively, which were equal or close to those observed when a mixture of Leishmania antigen (F45) was used (0.944). Moreover, F-CPB presented higher specificity and predictive positive value (0.955 and 0.980, respectively) than its domains (0.909 and 0.962 and 0.913 and 0.962 for the N-CPB and C-CPB, respectively) (Table 1). Overall, these results endorse F-CPB and its domains as effective tools in the diagnosis of CL in dogs, with high sensitivity and specificity.

TABLE 1.

Statistic parameters of the ELISA against the cysteine proteinase B from L. braziliensis (CPB) and its domains for the diagnosis of CL in dogs

Statistica Value for antigen
F45 F-CPB N-CPB C-CPB
Se 0.944 0.907 0.944 0.943
Sp 0.909 0.955 0.909 0.913
TP 51 49 51 50
FP 2 1 2 2
TN 20 21 20 21
FN 3 5 3 3
PPV 0.870 0.980 0.962 0.962
NPV 0.962 0.808 0.870 0.875
AUC 0.972 0.972 0.956 0.948
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a

Se, sensitivity; Sp, specificity; TP, true positive; FP, false positive; TN, true negative; FN, false negative; PPV, positive predictive value; NPV, negative predictive value; AUC, area under the curve.

Based on a thorough analysis of clinical and epidemiological data, CL dogs were then subdivided as follows: A, dogs bearing ulcerative lesions typical of CL; B, dogs without ulcers, living in the houses of humans or other dogs with leishmaniasis; C, dogs with atypical ulcers, living in the houses of humans with leishmaniasis; D, asymptomatic dogs living in houses with human or other dogs without leishmaniasis. Interestingly, specific antibodies against all the antigens tested were significantly higher in groups A, B, and C, which corresponded to dogs that had or could have been exposed to Leishmania parasites, than in asymptomatic dogs (group D). Titers of specific antibodies against the recombinant proteins agreed with those observed against the parasite lysate (Fig. 4).

FIG 4.

FIG 4

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ELISAs of sera from dogs living in Northwest Argentina. Sera were assayed for the presence of IgG antibodies against L. braziliensis promastigote lysate (F45), full-length CPB, and N- and C-terminal domains. Groups: A, dogs bearing ulcerative lesions typical of CL; B, dogs without ulcers, but living in the houses of humans or other dogs with leishmaniasis; C, dogs with atypical ulcers, living in the houses of humans with leishmaniasis; D, asymptomatic dogs from areas of endemicity living in houses with human or other dogs without leishmaniasis. Results are expressed as the titers of specific antibodies. Titers were calculated as the dilution in which the optical density (OD) obtained was equal to the mean of controls ± 2.23 SD for each antigen. **, P < 0.01; ***, P < 0 0.005; ****, P < 0.0001.

CPB and its domains in the diagnosis of VL.

We then analyzed the efficiency of the different antigens in the diagnosis of VL in dogs. As shown in Fig. 5, significant differences in reactivity against F-CPB and its domains were observed among dogs suffering from VL or not.

FIG 5.

FIG 5

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CPB and its domains in the diagnosis of canine visceral leishmaniasis. Sera from dogs from the northeast and center of Argentina were assayed for the presence of IgG antibodies against L. braziliensis promastigote lysate (F45), full-length CPB, and N- and C-terminal domains. Results are expressed as OD490. Lines represent the means ± SEMs. The cutoff (CO) for the different antigens was determined using the ROC curve. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

The ELISA containing F-CPB exhibited the best performance compared to the other antigens tested (AUC: 0.879, 0.789, and 0.723 for F-CPB, N-CPB, and C-CPB, respectively). These results mean that F-CPB as a coating antigen in an ELISA is a good candidate for the diagnosis of VL in dogs (Fig. 6). In addition, we observed higher sensitivity (Se) (93.3%) and specificity (Sp) (92.30%) for F-CPB than for the N- (Se, 73.3%; Sp, 76.9%) and C-terminal domains (Se, 66.7%; Sp, 88.5%) (Table 2).

FIG 6.

FIG 6

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Diagnostic efficacy of the recombinant antigens in canine VL using ROC curves. Sera from dogs from the northeast and center of Argentina were analyzed in an ELISA matrix against L. braziliensis: (A) promastigote lysate (F45), (B) full-length CPB, (C) N-terminal domain of CPB, (D) C-terminal domain of the CPB. True-positive rate (sensitivity) was plotted as a function of the false-positive rate (100% specificity) for the different Leishmania antigens at different cutoff points. An area of 1 represents a perfect test, while an area of 0.5 represents a worthless test. The accuracy of a diagnostic test is as follows: 0.90 to 1, excellent; 0.80 to 0.90; good; 0.70 to 0.80, fair; 0.60 to 0.70, poor; 0.50 to 0.60, fail.

TABLE 2.

Statistic parameters of the ELISA against cysteine proteinase B from L. braziliensis (CPB) and its domains for the diagnosis of VL in dogs

Statistica Value for antigen
F45 F-CPB N-CPB C-CPB
Se 0.867 0.933 0.733 0.667
Sp 1.000 0.923 0.769 0.885
TP 13 14 11 10
FP 0 2 6 3
TN 26 24 20 23
FN 2 1 4 5
PPV 1.0 0.875 0.647 0.769
NPV 0.929 0.960 0.833 0.821
AUC 0.941 0.879 0.7897 0.723
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a

Se, sensitivity; Sp, specificity; TP, true positive; FP, false positive; TN, true negative; FN, false negative; PPV, positive predictive value; NPV, negative predictive value; AUC, area under the curve.

The CPB amino acid sequence is highly conserved among Leishmania species.

To further analyze whether CPB might be a promising antigen for the diagnosis of leishmaniasis caused by the infection of several species, the amino acid sequence of the CPB from L. braziliensis was aligned with its orthologous sequences in different Leishmania species. As shown in Fig. 7, high percentages of identity were found: 91.5% for L. guyanensis (ACS66748.1), 76.1% for L. panamensis (ABX74953.1), 68.1% for L. major (XP_001681135.1), 62.8% for L. infantum (SUZ39418.1), 62.5% for L. donovani (AGI92544.1), 62.2% for L. mexicana (CAA90236.1), 61.5% for L. tropica (AFN27127.1), and 61.5% for L. aethiopica (AAZ23596.1). These results suggest that the CPB from L. braziliensis qualifies as a good target for the diagnosis of Leishmania species infection caused by different species of the parasite However, an exhaustive study of the ELISA performance of the CPB of L. braziliensis in Leishmania infections caused by all the mentioned strains should be carried out in the near future.

FIG 7.

FIG 7

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Conservation of the amino acid sequences of cysteine proteinase B (CPB) in different Leishmania species. (A) Alignment of the CPB from L. braziliensis with its orthologous sequences from L. guyanensis (ACS66748.1), L. panamensis (ABX74953.1), L. major (XP_001681135.1), L. infantum (SUZ39418.1), L. donovani (AGI92544.1), L. mexicana (CAA90236.1), L. tropica (AFN27127.1), and L. aethiopica (AAZ23596.1). (B) Phylogenetic tree based on the amino acid sequence of the CPB in Leishmania.

DISCUSSION

A rapid and accurate diagnosis of Leishmania species infection followed by the early implementation of an effective treatment in infected individuals is essential for the control of a disease that has spread for several reasons. Domestic dogs are considered the main reservoirs of L. infantum, playing an important role in the epidemiology of VL (42, 43). The number of infected dogs in South America is estimated in the millions, and there are high infection rates associated with a high risk of human disease (42–44). Although the development of sensitive molecular diagnostic techniques has improved the detection of clinically healthy infected dogs, those methods are not always available to researchers in Latin America.

Immunoserological tests have evolved as useful tools in the diagnosis of leishmaniasis in dogs, since the humoral response in general is intense, with high levels of specific immunoglobulins (45–47). We showed in an ELISA that the CPB from L. braziliensis and its domains, mainly F-CPB, are promising antigens for the diagnosis of both cutaneous and visceral clinical presentations of leishmaniasis in dogs, with high sensitivity and specificity (Se, 90.7; Sp, 95.5; AUC, 0.97; and Se, 93.3; Sp, 92.3; AUC, 0.88, respectively) (Fig. 2 and 5 and Tables 1 and 2). Moreover, the high sensitivity of the CPB from L. braziliensis in the diagnosis of VL (93.3%) could be explained considering the higher stimulation of the immune system in the visceral form compared to that with a localized cutaneous presentation (Se, 90.7). In that regard, several reports (48–50) have shown the importance of the CPBs from L. infantum and L. (L.) chagasi as targets of the humoral and cellular immune response and their potential use for the diagnosis of VL in humans and dogs.

Bearing in mind that the species that cause CL and VL disease are generally different, the ability of the CPB from L. braziliensis to detect the infection caused by different Leishmania strains highlights its value as a candidate for the universal diagnosis of leishmaniasis. This is also supported by the conserved amino acid sequence of this antigen among several Leishmania species (Fig. 7).

One limitation of most serological tests is their inefficiency for detecting VL in dogs during the early stages of infection. Early detection of canine VL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In that regard, Faria et al. (51) reported an ELISA for two multiepitope proteins, PQ10 and PQ20, which was able to detect Leishmania infection at earlier time points than with kinetoplast DNA (kDNA) PCR-restriction fragment length polymorphism (RFLP) in anti-IgG and anti-IgM assays. As shown in Fig. 4, we observed that dogs without ulcers living in contact with humans with leishmaniasis (group B) displayed a significant increase in IgG titers against F-CPB and its domains compared with that in asymptomatic dogs (group D). These results indicate that the CPB of L. braziliensis can be a good predictor of Leishmania species infection yielding significant serum IgG antibodies in the host before the onset of leishmaniasis symptoms. This hypothesis needs to be further explored in future studies.

Recently, Lima et al. (52) showed the high sensitivity and specificity of an ELISA from an L. braziliensis kinesin-like hypothetical protein (LbHyM) for the serodiagnosis of human cutaneous and mucosal leishmaniasis. Nearly 78% similarity was found in the amino acid sequence comparison between LbHyM and the T. cruzi hypothetical protein. The strong cross-reactivity between Leishmania and T. cruzi makes their differential serodiagnosis difficult. Since the drugs used for the treatment of both parasitoses are different, an accurate diagnosis is necessary. In a preliminary study, we recently observed no cross-reactivity between T. cruzi-infected patients and the CPB of L. braziliensis by ELISA. Additionally, sera from patients that were positive for the F-CPB from L. braziliensis and its domains did not recognize T. cruzi-specific antigens, such as cruzipain, thiol-transferase (Tc52), and the flagellar calcium-binding protein (Tc24), in an immunoblotting assay. In contrast, all these T. cruzi antigens were recognized in samples from patients with Chagas disease (data not shown).

We conclude that the performance of the CPB from L. braziliensis and its domains turns them into promising antigens for the diagnosis of leishmaniasis in dogs caused by different Leishmania species. Furthermore, it must be considered that the ELISA, with potential application in areas of endemicity, could be further improved by the addition of other antigens or the use of different blocking reagents or different detection systems, such as streptavidin-peroxidase. The analysis of potential cross-reactivity with other coendemic diseases and pathogens must be further investigated as the next step to validate CPB in the diagnosis of Leishmania species infection.

ACKNOWLEDGMENTS

This work was supported by grants from Agencia Nacional de Promoción Científica y Técnica (PICT number 00608) and from Universidad de Buenos Aires, Argentina (20020100200160, 20020090200457).

We thank Diego Eiras for his kind contribution to the collection of dog sera.

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