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Journal of Clinical Microbiology, November 2005, p. 5515-5519, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5515-5519.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of Enzyme-Linked Immunosorbent Assays, an Immunofluorescent-Antibody Test, and Two Rapid Tests (Immunochromatographic-Dipstick and Gel Tests) for Serological Diagnosis of Symptomatic and Asymptomatic Leishmania Infections in Dogs

Maik Mettler,¹ Felix Grimm,¹ Gioia Capelli,² Heinrich Camp,³ and Peter Deplazes^1*

Institute of Parasitology, University of Zurich, Zurich, Switzerland,¹ Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy,² Veterinary Clinic "Am Forstgarten," Kleve, Germany³

Received 4 May 2005/ Returned for modification 11 June 2005/ Accepted 13 August 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enzyme-linked immunosorbent assays (ELISAs) based on soluble antigens derived from promastigote or amastigote-like stages of Leishmania infantum and on the recombinant rK39 antigen, each in combination with different conjugates [anti-immunoglobulin G1 [IgG1], anti-IgG2, anti-IgG(), and anti-IgG heavy plus light chains], were compared to an immunofluorescent-antibody test (IFAT) and two commercially available rapid test systems (DiaMed-Vet-IT Leish and ID-PaGIA canine leishmaniasis antibody test) for the detection of specific anti-Leishmania antibodies in symptomatic and asymptomatic dogs with proven L. infantum infections. ELISAs based on soluble promastigote and amastigote antigens had very high sensitivities in symptomatic (n = 30; 100%) and asymptomatic dogs (n = 17; 94.1 to 100%), except when combined with the anti-IgG1 conjugate (41.2 to 82.4%). Specificities were high for all combinations (n = 50; 96 to 100%). The rK39 ELISA detected fewer asymptomatic cases (sensitivities, 52.9 to 64.7%) but was highly specific (96 to 100%). The IFAT was 90% sensitive in symptomatic dogs but was significantly less sensitive in asymptomatic cases (29.4%). However, it had an excellent specificity (100%). Test performances of the rapid tests based on the rK39 antigen were comparable to the ELISAs based on the same antigen. ELISAs based on soluble promastigote or amastigote antigens seem to be most suited for the serological diagnosis of canine Leishmania infections in both symptomatic and asymptomatic dogs. IFAT and the rK39 ELISA lack sensitivity in asymptomatic cases but are highly specific. Rapid tests like the rK39 dipstick test or the ID-PaGIA are helpful for confirming clinically suspected cases because of their high specificities in symptomatic animals.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In countries around the Mediterranean basin, human visceral leishmaniasis and canine leishmaniasis are endemic. Both diseases are caused by Leishmania infantum, and dogs are the main reservoir of the parasite. In some areas, prevalences of canine leishmaniasis vary between 1 and 37% (7). Canine leishmaniasis is also endemic to Latin America, where it is caused by Leishmania chagasi, now considered synonymous with L. infantum (3). Leishmaniasis is one of the most important imported canine diseases in Central Europe (23). Leishmania infections have also recently been found in foxhounds in the United States (14), where the parasite is likely to become endemic (12).

The clinical diagnosis of canine leishmaniasis is difficult due to the variable symptomatology. The definite diagnosis often depends on parasite isolation by in vitro culture or by detection of parasite DNA by PCR from lymph node or bone marrow biopsy specimens (22). However, these techniques are invasive, time-consuming, and expensive. Microscopic demonstration of the parasite in Giemsa-stained smears of lymph node or bone marrow aspirates lacks sensitivity (17) and is not considered adequate for diagnosis of canine leishmaniasis. Thus, the detection of specific anti-Leishmania antibodies in canine sera remains an important diagnostic tool. Among the different tests available, most widely used are immunofluorescent-antibody tests (IFAT) (21), direct agglutination tests (19), enzyme-linked immunosorbent assays (ELISAs) (5), dot-ELISAs (13), and Western blots (1, 15). These tests are mostly based on purified, water-soluble antigen fractions of promastigote or amastigote stages or on a recombinant antigen (rK39) containing a repetitive, immunodominant epitope of a kinesin-related protein that is highly conserved among viscerotropic Leishmania species (9).

Symptomatic dogs usually produce high levels of specific antibodies, particularly immunoglobulin G1 (IgG1) and IgG2, that can easily be detected (10). However, the sensitivity of antibody detection is generally lower in early or in asymptomatic canine infections (20).

In the present study, we evaluated ELISAs using rK39, promastigote, and amastigote antigens in combination with various conjugates and compared them with a commonly used IFAT and with two commercially available rapid test systems for the detection of specific anti-Leishmania antibodies in symptomatic and asymptomatic dogs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dogs and infection status. Group 1 contained serum samples from 48 stray or abandoned dogs from a region of nonendemicity in Southern Switzerland (11). These sera were solely used to determine the positive-negative thresholds of the different ELISAs (see below). Group 2 contained serum samples from 47 Leishmania-infected dogs (17 clinically asymptomatic and 30 symptomatic) originating or returning from Mediterranean areas. Infections were proven by in vitro cultivation and/or PCR of lymph node aspirates as described by Mathis and Deplazes (22). Group 3 contained serum samples from 50 healthy blood donor dogs collected at the Clinic for Small Animal Internal Medicine, Veterinary Faculty, University of Zurich. Group 4 contained serum samples from 26 dogs with other parasite infections, as follows: Babesia canis, n = 7, parasites present in Giemsa-stained blood smears; Toxoplasma gondii, n = 8, seropositive by ELISA; Neospora caninum, n = 9, seropositive by IFAT; Hepatozoon canis, n = 2, parasites present in Giemsa-stained blood smears.

ELISA. Promastigote and amastigote-like stages of L. infantum (MCAN/ES/89/IPZ229/1/89; zymodeme MON-1) were grown in axenic in vitro cultures (4, 18). Parasites were washed three times in phosphate-buffered saline (PBS) by centrifugation (1,500 x g, 15 min, 8°C), repeatedly (5 cycles) frozen in liquid nitrogen and thawed in a 37°C water bath, ultrasonicated (twice for 2 min), and finally centrifuged at 15,000 x g for 60 min (4°C). The supernatant was dialyzed at 4°C overnight against PBS and stored in aliquots at –80°C. Protein concentrations were estimated by the Bio-Rad (Hercules, CA) protein assay with bovine albumin as the standard. The recombinant antigen rK39 was kindly provided by DiaMed AG (Cressier sur Morat, Switzerland).

All ELISA procedures were optimized with regard to antigen concentrations and conjugate dilutions (data not shown). Positive-control sera from dogs with parasitologically proven infections and negative-control sera from specific-pathogen-free dogs were included in all tests. Soluble antigens were diluted in 0.1 M carbonate-bicarbonate buffer (pH 9.6) containing 0.02% NaN₃. Optimal dilutions were 1:100 for the promastigote (2.5 µg/ml), 1:200 for the amastigote (2.5 µg/ml), and 1:5,000 for the rK39 (0.2 µg/ml) antigens, respectively. One hundred microliters per well was used to coat 96-well microtiter plates (Nunc Maxisorp, Roskilde, Denmark) overnight at 4°C. Plates were washed three times with 0.9% NaCl-0.3% (vol/vol) Tween 20 and saturated for 30 min at 37°C with PBS (pH 7.2) containing 0.02% NaN₃, 0.05% bovine hemoglobin (Fluka, Buchs, Switzerland), and 0.3% (vol/vol) Tween 20 (PBS-T). Dog sera were diluted 1:200 in PBS-T, and 100 µl per well was incubated for 1 h at 37°C. Plates were washed four times, and 100 µl of the diluted detection antibodies was added per well. Four different secondary antibodies, conjugated to alkaline phosphatase (conjugates), were diluted in PBS-Tween 20: goat anti-dog IgG(),1:1,000 (Kirkegaard and Perry Lab, Inc., Gaithersburg, MD); sheep anti-dog IgG2, 1:1,600; goat anti-dog IgG heavy plus light chains (H+L), 1:8,000; goat anti-dog IgG1, 1:100 (Bethyl Laboratories, Inc., Montgomery, TX). After 1 h at 37°C, the plates were washed four times, and 100 µl of a 1-mg/ml solution of p-nitrophenyl phosphate (Sigma) in 0.05 M carbonate-bicarbonate buffer containing 1 mM MgCl₂ (pH 9.8) was added to each well. After 20 to 30 min, reactions were stopped by adding 50 µl of 3 M NaOH to each well. Absorbance was measured at 405 nm (A₄₀₅; reference filter, 630 nm) in a Multiskan EX microplate reader (Thermo Labsystems, Helsinki, Finland). Samples were considered positive if the A₄₀₅ value was above the arithmetic mean plus 3 standard deviations of the A₄₀₅ values of 48 negative controls (sera of group 1).

IFAT. IFAT was performed at the Istituto Zooprofilattico Sperimentale delle Venezie (Padua, Italy), as previously described by Gradoni and Gramiccia (16), using promastigotes of L. infantum zymodeme MON-1 as the antigen. Anti-Leishmania antibodies were detected using rabbit anti-dog IgG (whole molecule) secondary antibodies conjugated to fluorescein isothiocyanate (Sigma-Aldrich, Inc., Saint Louis, MO). Samples were classified positive if promastigote cytoplasmatic or membrane fluorescence was observed at a serum dilution of 1:40 or higher.

Rapid tests. An immunochromatographic-dipstick test (DiaMed-Vet-IT Leish) and a gel test (ID-PaGIA Canine leishmaniasis antibody test; Diamed AG, Cressier sur Morat, Switzerland) were used according to the manufacturer's instructions. Both tests were based on the rK39 antigen.

Test parameters and statistics. Test sensitivities were calculated for the asymptomatic and symptomatic dogs of group 2 separately as percentages of test-positive sera per true positives (17 in the asymptomatic subgroup and 30 in the symptomatic subgroup). Test specificities are based on dog sera from group 3 (healthy dogs from a region of nonendemicity, n = 50) and were calculated as percentages of test-negative sera per true negatives. Cross-reactions are given as percentages of positive results among group 4 sera. The test parameters of the different ELISAs and the two rapid tests were compared to the corresponding parameters of IFAT, which is considered the gold standard for serological diagnosis of canine leishmaniasis in many regions (17). Differences among test parameters were statistically analyzed by the Cochran Q test (Analyze-it; Analyze-it Software Ltd., Leeds, United Kingdom).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Table 1 summarizes the results of the different diagnostic tests used in this comparative study.

View this table: [in this window] [in a new window]

TABLE 1. Sensitivities and specificities of ELISAs, IFAT, and two rapid tests for detection of anti-Leishmania antibodies in canine seraa

Sensitivities. The ELISAs based on promastigote or amastigote antigens detected specific anti-Leishmania antibodies in all symptomatic dogs (n = 30) and in 16 to 17 of 17 asymptomatic dogs with parasitological evidence of Leishmania infections when used in combination with IgG()-, IgG2-, or IgG H+L-specific secondary antibodies (sensitivities, 94.1 to 100%, differences not statistically significant). On the contrary, the sensitivities were significantly (P < 0.01) lower (33.3% and 41.2% in symptomatic and asymptomatic cases, respectively) when promastigote antigen and IgG1-specific detection antibodies were used. These discrepancies were less pronounced for amastigote-derived antigens, with corresponding sensitivities of 86.7% (not significant) and 82.4% (marginally significant, P = 0.045), respectively.

ELISAs based on the rK39 antigen performed well in symptomatic cases [sensitivities of 96.7% for IgG()-, IgG2-, and IgG H+L-specific detection antibodies]. Again, the sensitivity was significantly lower (80%, P < 0.05) for the IgG1-specific detection antibodies. In asymptomatic dogs, sensitivities were significantly (P < 0.01) lower than the promastigote and amastigote antigen-based ELISAs and ranged from 52.9 to 64.7%, depending on the conjugate used.

IFAT was 90% sensitive for symptomatic cases but missed 12 of 17 infected dogs without clinical signs of leishmaniasis, resulting in decreased sensitivity (29.4%) for asymptomatic cases.

The rapid tests were comparable to the ELISAs in symptomatic cases with sensitivities of 96.7% and 100%, respectively (differences not significant). In asymptomatic dogs, the dipstick test was significantly less sensitive and detected 9 of 17 cases (52.9%, P < 0.01), whereas 13 of the 17 asymptomatic dogs were found positive by the gel test (76.5%, P < 0.05).

For the detection of asymptomatic cases, ELISAs based on promastigote (except in combination with the IgG1-specific conjugate) and amastigote antigens and the ID-PaGIA performed significantly better than the widely used IFAT (P < 0.01) (Table 1).

Specificities. The specificities, as assessed with sera from healthy blood donor dogs from Switzerland (n = 50, group 3), ranged from 96 to 100% in the ELISAs and from 92 to 94% in the rapid tests, respectively. All ELISAs were 100% specific when IgG1-specific secondary antibodies were used. No false-positive results were recorded with the IFAT (specificity, 100%). Except for the ID-PaGIA, which showed a marginally lower specificity (92%, P = 0.045), the observed minor differences within this group were not statistically significant.

Cross-reactions (group 4, n = 26). In the ELISAs based on promastigote or amastigote antigens, cross-reactions were observed in dogs infected with Babesia canis, Toxoplasma gondii, Neospora caninum, or Hepatozoon canis in 1 to 10 cases from 26 dogs, depending on the conjugate used (3.8% to 38.5% cross-reactions). The highest numbers of false-positive results were observed for the IgG H+L-specific secondary antibodies. Except for the IgG1-specific conjugate, which yielded three false-positive results, all 26 dogs with other parasitic infections were negative by the rK39 ELISA. In both the rK39 dipstick test and the gel test, one single cross-reaction was noted (Neospora caninum and Hepatozoon canis, respectively; 4.3% and 3.8%, respectively). It has to be noted that, in the latter, cross-reactions were assessed with only 23 sera because three samples that gave doubtful results were excluded. Such results might be related to the quality of the samples, and according to the manufacturer's instructions, should not be considered valid. Again, no false-positive IFAT result was recorded in this group of sera. However, the differences to the other tests were not significant or only marginally significant in most cases (Table 1).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The requirements of a serological test for canine leishmaniasis may vary. For confirmatory purposes of clinically suspected cases, test specificity is most important, whereas a high sensitivity is most essential in surveillance programs or for testing of dogs imported from areas of endemicity to detect asymptomatic carriers. Such animals are a potential source of infection for sandfly vectors (2), and the probability of developing overt disease within 1 year is higher than 30% (25).

In our study, all ELISAs based on soluble promastigote and amastigote antigens in combination with IgG()-, IgG2-, and IgG H+L-specific detection antibodies had very high sensitivities in symptomatic (100%) and asymptomatic (94.1 to 100%) dogs. In contrast, in combination with the IgG1-specific conjugate, the sensitivities were lower for both groups of dogs. This decrease was statistically significant (P < 0.01) for promastigote-derived antigens but less pronounced and not significant for amastigote antigens. The rK39 ELISAs did not vary significantly with respect to sensitivity and specificity for the different conjugates. However, they detected considerably fewer asymptomatic cases than tests based on soluble promastigote- or amastigote-derived antigens.

Although specific antibodies of both the IgG1 and IgG2 subclasses occur in naturally infected dogs, IgG2 antibodies seem to predominate (8, 10). This might explain the decreased sensitivities in tests using IgG1-specific conjugates. This lower sensitivity was particularly observed in tests with crude promastigote antigens but was much less pronounced for the rK39 and amastigote antigens.

Not surprisingly, the highest numbers (6 with promastigote antigens and 10 with amastigote antigens, respectively) of false-positive results occurred when IgG H+L detection antibodies were used. These recognize IgG1 and IgG2 antibodies as well as light chains common to other immunoglobulins and were used together with crude antigen fractions that offer a mixture of different epitopes. Consistently, this observation was not observed for the combination of the IgG H+L conjugate and the highly defined rK39 antigen. The results demonstrate that both antigens and detection antibodies, as well as their combinations, are decisive for determining the diagnostic features of specific antibody detection in canine leishmaniasis.

Various studies of human patients with visceral leishmaniasis and of dogs with canine leishmaniasis have demonstrated that rK39-based ELISAs are sensitive (93 to 100%) and specific (94 to 100%) (6, 28, 29). Furthermore, the presence of specific anti-rK39 antibodies seems to be correlated to some extent with active disease in humans and dogs (6, 27). The present findings support these results. Specific anti-rK39 antibodies were detected in 96.7% of dogs with apparent disease (only 80% when using IgG1) but only in 52.9 to 64.7% of asymptomatic animals.

In most countries around the Mediterranean basin, IFAT is considered the gold standard for serological diagnosis of canine leishmaniasis, and it is generally recognized as the most sensitive and specific test (17). In our study, IFAT was 90% sensitive in symptomatic dogs, but anti-Leishmania antibodies could be detected in only 5 of 17 asymptomatic dogs with proven infections. Its sensitivity (29.4%) in this group of dogs was significantly lower than those of the ELISAs using promastigote- or amastigote-derived antigens. However, IFAT was outstanding with its excellent overall specificity of 100%, as assessed with healthy dogs and with dogs with other parasitic infections.

Dipstick tests have various basic advantages compared to the other test formats. They are ready for use, individually packed, easy to handle, and rapid. Additionally, the DiaMed-Vet-IT Leish assay (dipstick) may be stored at ambient temperature (up to 30°C) and is suitable for field use. The test is based on the rK39 antigen, and its sensitivity and specificity were comparable to those of the rK39-based ELISAs (no significant differences) and were within the ranges reported in earlier evaluations of other rK39-based dipstick assays (24, 26).

The advantage of the ID-PaGIA leishmaniasis gel test over other rapid tests is the large number of samples that can be examined in one working step (up to 144). A test run is completed in 20 min, and as with dipstick assays, results can be read by the naked eye. However, according to the manufacturer, old or lipemic samples or sera that have been repeatedly frozen and thawed may lead to erroneous results and should not be used. In addition to a 100% detection rate for symptomatic dogs, the ID-PaGIA also detected 76.5% of asymptomatic cases. The numbers of false-positive results were not significantly different from those of the other assays.

ELISAs based on soluble promastigote or amastigote antigens are well suited for the serological diagnosis of canine leishmaniasis in both symptomatic and asymptomatic dogs. For practical reasons, promastigote-derived antigens are preferable because these stages are easier to maintain in culture systems. IgG()- and IgG2-specific conjugates can be recommended as detection antibodies, whereas IgG1-specific conjugates are not suitable.

The rK39 ELISA evaluated in the present study and the IFAT lack sensitivity for the detection of asymptomatic dogs, but they are highly specific. Rapid tests like the DiaMed-Vet-IT Leish (dipstick test) and the ID-PaGIA Canine leishmaniasis antibody test are helpful for confirming clinically suspected cases because of their high specificity in symptomatic animals.

 
The rapid tests were kindly supplied by Diamed AG, Cressier sur Morat, Switzerland.

We thank Dominik Heinzmann for help with the statistical analyses.

 
* Corresponding author. Mailing address: Institute of Parasitology, University of Zurich, Winterthurerstr 266a, CH-8057 Zurich, Switzerland. Phone: 41 44 635 8502. Fax: 41 44 635 8907. E-mail: deplazesp{at}access.unizh.ch.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, November 2005, p. 5515-5519, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5515-5519.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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• Falqueto, A., Ferreira, A. L., dos Santos, C. B., Porrozzi, R., da Costa, M. V. S., Teva, A., Cupolillo, E., Campos-Neto, A., Grimaldi, G. Jr (2009). Cross-sectional and Longitudinal Epidemiologic Surveys of Human and Canine Leishmania infantum Visceral Infections in an Endemic Rural Area of Southeast Brazil (Pancas, Espirito Santo). Am J Trop Med Hyg 80: 559-565 [Abstract] [Full Text]  
• Otranto, D., Paradies, P., de Caprariis, D., Stanneck, D., Testini, G., Grimm, F., Deplazes, P., Capelli, G. (2009). Toward Diagnosing Leishmania infantum Infection in Asymptomatic Dogs in an Area Where Leishmaniasis Is Endemic. CVI 16: 337-343 [Abstract] [Full Text]  
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