An Iron-Superoxide Dismutase Antigen-Based Serological Screening of Dogs Indicates Their Potential Role in the Transmission of Cutaneous Leishmaniasis and Trypanosomiasis in Yucatan,Mexico

Abstract

An increasing number of studies have reported high infection rates for American cutaneous leishmaniasis in dogs, which have thus been proposed as the reservoir host. Canine leishmaniasis is widespread in different states in Mexico, where a number of Leishmania species have been isolated from dogs. In the present study, the detection of different Leishmania species is described in stray dogs from two localities, namely Tulum and Celestún on the Yucatan Peninsula (Mexico). The use of iron-superoxide dismutase excreted by the parasites as the antigen fraction and enzyme-linked immunosorbent assay and western blot tests allowed us to confirm the presence of at least three species of Leishmania (Le. mexicana, Le. braziliensis, and Le. panamensis), some of which are reported for the first time in this species. In addition to a high prevalence of Le. mexicana and Le. braziliensis, and to a lesser degree, Le. panamensis, there is a significant prevalence of Trypanosoma cruzi, suggesting that the dog may be a source of transmission of trypanosomiasis. However, a more thorough epidemiological study on the dog population, both wild as well as urban, of the Yucatan Peninsula will be required to design a control strategy for these diseases, paying particular attention to the population affected and even broadening the study to other Mexican states as well as neighboring countries. These results again confirm that iron-superoxide dismutase excreted by the different trypanosomatid species constitutes a good source of antigen for serodiagnosis in epidemiological studies.

Introduction

T he term leishmaniasis covers a group of diseases transmitted by the sand fly and caused by kinetoplastids belonging to the genus Leishmania. These parasitoses are widely distributed all over the world except Antarctica (www.who.int/Leishmaniasis/Leishmaniasis_maps/en/index.html). In recent years, leishmaniasis has begun to be considered a group of emerging diseases as a result of climate change, population migration, the resistance of mosquitoes to insecticides, and the difficulty of eliminating the infection in reservoir animals (Ameen 2010).

Leishmaniasis is endemic in 88 countries, and according to WHO estimates, 12 million people are currently infected, 350 million are at risk, and some 2 million new cases are reported each year. Thus, the disease constitutes a serious public health problem because of its significant economic, social, and psychological costs. However, these figures are likely to be an underestimate, as reporting of the disease is only compulsory in 33 countries, which means that most cases go unreported (WHO 2008).

New World leishmaniasis is currently distributed from the south of Texas to Argentina, with only Chile, Uruguay, and the Caribbean currently being leishmaniasis free. Cases of leishmaniasis have been reported in 20 states in Mexico, where the main endemic regions are Campeche, Chiapas, Yucatán, Oaxaca, Quintana Roo, Tabasco, and Veracruz (Bonfante-Garrido 1983, Córdova-Uscanga et al. 1993, Velasco Castrejon et al. 2009, Sánchez-García et al. 2010). The most common clinical manifestation in these states is local cutaneous leishmaniasis, whose transmission cycle involves different species of Lutzomyia (Lu. olmeca, Lu. whitmanni, Lu. pessoai, Lu. mogonei, and Lu. intermedia, amongst others) (Bonfante-Garrido 1983).

Leishmaniasis was first reported in the Yucatan Peninsula (Mexico) in 1912 (Seidalin 1912). In some areas, especially in “Chiclero's Ulcer” regions, cutaneous and mucocutaneous leishmaniasis are considered endemic, as demonstrated by various studies undertaken since the 1950s (Rebollar-Téllez et al. 1996, Andrade-Narváez et al. 2003, 2005). However, a major change observed in the ecosystem recently could be altering the transmission dynamics in both vectors and reservoirs, as suggested by studies undertaken in Chetumal (Quintana Roo) and Tabasco (Córdova-Uscanga et al. 1993, Sánchez-García et al. 2010).

Although rodents have always been considered to be the main reservoirs of this disease, the discovery of the first dog infected with Leishmania sp. in Brazil in 1913 suggested the possibility that dogs play a much more important role in the transmission of this disease on a global scale as a result of their movement between wild and domestic environments (Pedroso 1913). Indeed, numerous new cases of canine leishmaniasis have been reported in many countries recently (Marco et al. 2005, Castro et al. 2007, Dantas-Torres 2007, Hotez 2008). The first report concerning dogs infected by Leishmania mexicana in the states of Campeche, Quintana Roo, and Oaxaca appeared in 2009 (Velasco Castrejon et al. 2009). In light of these reports, it has been proposed that the dog could be one of the main reservoirs involved in transmission of this disease in a periurban and urban context and that, because of its synanthropy, it could be a vehicle of transmission from the wild to an urban setting.

Leishmaniasis is difficult to diagnose because of its broad spectrum of clinical manifestations, many of which are nonspecific, and the type of immunological response induced by the disease. The most common diagnostic methods include direct visual detection of the parasite, often in combination with immunological or molecular techniques (Reithinger and Dujardin 2007) such as the Montenegro reaction (for cutaneous leishmaniasis) or indirect fluorescent antibody test, direct agglutination test, enzyme-linked immunosorbent assay (ELISA), and western blot (or visceral leishmaniasis), all of which use whole parasite extract or the whole parasite (Reale et al. 1999). Other (nonconventional) serological tests use recombinant proteins, such as Hsp70, Hsp90, Leishmania donovani A2 antigen, and the recombinant protein rK26 or rK39, as the antigen in an immunochromatographic dipstick (Gomes et al. 2008). However, the effectiveness of using whole-parasite extract in such serological assays is somewhat limited because of the dubious reproducibility and specificity of the results obtained, especially in regions where other parasites that can give a cross-reaction, such as Trypanosoma cruzi, coexist (Chiller et al. 1990, Marín et al. 2009).

Many studies aimed at discovering a Leishmania-specific antigen that could increase the specificity of serodiagnosis have been performed (Mohapatra et al., 2010, Rasouli et al., 2009, Saridomichelakis 2009). One such candidate antigen is excreted iron-superoxide dismutase (Fe-SODe), which has been found to be highly immunogenic and specific, thus making it a useful molecular marker for diagnosing infection with these parasites. Indeed, this antigen has been shown to provide good results in the diagnosis of visceral canine leishmaniasis (Marín et al. 2007) and both cutaneous and mucocutaneous leishmaniasis in human serum in Peru (Marín et al. 2009).

The chief aim of the present work was to develop an ELISA using antigens (Fe-SODe) excreted by Le. mexicana, Leishmania braziliensis, and Leishmania panamensis as a basis for the development of a serodiagnostic tool. Further, as a test of this methodology, we describe the percentage of stray dogs infected with these parasites in the localities of Tulum and Celestun on the Yucatan Peninsula (Mexico). Likewise, we demonstrate the lack of cross-reactions between the different Leishmania spp. and other trypanosomatids such as T. cruzi. The use of Fe-SODe could have great diagnostic and epidemiological value for both canine leishmaniasis and other parasitic diseases caused by different species of the genus Leishmania.

Materials and Methods

Parasites and culture

Promastigotes of Le. braziliensis (MHOM/BR/75/M2904), Le. mexicana (MHOM/BZ/82/Bel 21), Le. panamensis (MHOM/PA/71/LS94), and T. cruzi (MHOM/VE/1994/Maracay) were grown in axenic medium trypanosomes liquid (MTL; Gibco) supplemented with 10% heat-inactivated fetal bovine serum at 28°C in Falcon flasks.

Whole-parasite extract (fraction H)

The parasite culture (in the exponential growth phase) was concentrated by centrifugation at 1500 rpm for 10 min. The cell pellet was then washed twice with phosphate-buffered saline (PBS) and resuspended in ice-cold sodium Tris/HCl ethylenediamine tetraacetic acid (EDTA) buffer (0.25 M sucrose, 25 mM Tris/HCl, 1 mM EDTA, pH 7.8; buffer 1). The resulting pellet (0.5–0.6 g wet weight mL^–1) was suspended in 3 mL of buffer 1 and disrupted by three cycles of sonic disintegration (30 s each at 60 V). The sonicated homogenate was centrifuged at 1500 rpm for 10 min at 4°C, and then the pellet was washed three times with buffer 1 for a total supernatant fraction of 9 mL. This fraction was centrifuged (2500 rpm for 10 min at 4°C) and the supernatant (fraction H) was collected.

Extraction and purification of the SOD excreted (Fe-SODe)

Parasite forms in the exponential growth phase, obtained as described earlier, were concentrated by centrifugation at 1500 rpm for 10 min, the cell pellet was washed twice in serum-free MTL medium, then the number of cells was counted in an hemocytometric chamber, and the cells were distributed into aliquots of 5 × 10⁹ parasites/mL. Subsequently, the parasites were again grown in serum-free MTL medium for 24 h, the supernatant was collected by centrifugation at 1500 rpm for 10 min and then passed through a filter of 0.45-μm pore size, and solid ammonium sulfate was added. The protein fraction, which precipitated at a salt concentration of between 35% and 85%, was centrifuged (9000 rpm for 20 min at 4°C), redissolved in 2.5 mL of 20 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA (buffer 2), and dialyzed on a Sephadex G-25 column (Pharmacia, PD 10) previously balanced with buffer 2, to give a final volume of 2.5 mL (Fe-SODe fraction) (Marín et al. 2004).

The H and Fe-SODe fractions were both used as antigen fractions in ELISA and western blot assays. The protein content was determined using the Bio-Rad test, based on the Bradford method (Sigma Immunochemical), with bovine serum albumin as standard (Marín et al. 2007).

Serological assay (ELISA)

Fractions H and Fe-SODe from the parasites (Le. braziliensis, Le. mexicana, Le. panamensis, and T. cruzi), cultured and processed as described earlier, were used as the antigen fraction for the ELISA assay in all cases. The total homogenate (fraction H) and purified protein fraction (Fe-SODe), at a concentration of 5 and 1.5 μg, respectively, were coated onto polystyrene microtiter plates (Nunc) in carbonate buffer (pH 8.2) for 2 h at 37°C. The antigen remaining on the plate was eliminated by washing three times with PBS-Tween 20^® 0.05% (washing buffer). Free adsorption sites were blocked by incubation (2 h at 37°C) with blocking buffer (PBS-Tween 20 0.2%, bovine serum albumin 1%). The antibodies retained at a serum dilution of 1:200 in PBS were developed with peroxidase-labeled sheep anti-total-dog immunoglobulin antibodies at a dilution of 1:1000 as conjugate. The enzyme reaction was developed in the dark with the chromogenic substrate o-phenylenediamine dihydrochloride (Sigma^®, Madrid, Spain) and 10 μL of 30% H₂O₂ per 25 mL for 20 min. The reaction was stopped by addition of 50 μL of 3 N HCl, and the absorbance was read at 492 nm in a microplate reader (Metertech Σ 960). All samples were analyzed in triplicate in polystyrene microtiter plates, and the mean and standard deviations of the optical densities of the negative control sera (10 healthy dogs) were used to calculate the cutoff value (mean + 3 standard deviation).

Western blot analysis

The antigen fraction of Fe-SODe (at a concentration of 1.5 μg protein) from Le. braziliensi, Le. mexicana, Le. Panamensis, and T. cruzi was run on Isoelectric focusing (IEF) 3–9 gels and then transferred to nitrocellulose, for 30 min, as described in the Phast System manual. The membrane was blocked for 2 h at room temperature using 0.4% gelatin and 0.2% Tween 20 in PBS, then washed three times with 0.1% Tween 20 in PBS (PBS-T), and incubated for 2 h at room temperature with dog sera at a dilution of 1/200. Before washing again, the membrane was further incubated for 2 h at room temperature with a second antibody, namely anti-dog immunoglobulin G (Fc-specific) peroxidase conjugate (Sigma Immunochemical; dilution 1/1000). After washing as described earlier, the substrate diaminobenzidine (0.5 mg/mL in buffer Tris/HCl 0.1 M, pH 7.4, containing 1/5000 H₂O₂ [10 v/v]) was added and the reaction was stopped by washing several times with distilled water.

Study area

The study was carried out in Celestún, in the state of Yucatan, and Tulum, in the state of Quintana Roo (Mexico). The climate in this region is humid and subhumid tropical, with a summer rainy season. The monthly maximum temperatures vary from 25°C to 40°C, with a mean temperature of 26°C. The relative humidity varies from 60% to 95% (mean relative humidity: 80%), with an annual rainfall of 950 mm.

Dog population and sample collection

A total of 70 serum samples from stray dogs (55 from Celestún and 15 from Tulum) were obtained between 2008 and 2009. A 5 mL sample of whole blood was drawn from the cephalic vein of each dog into assay tubes (Vacuttainer; Beckton-Dickinson) and kept at 4°C. The negative control sera (10 healthy or asymptomatic dogs) were obtained from stray dogs put down by the veterinary services in Granada (Spain), which were not reactive to the western blot techniques.

Results

None of the 15 sera from Tulum showed a reaction against Leishmania sp. in the ELISA-H (whole-parasite extract as the antigen), whereas 8 sera tested positive for at least one species of cutaneous leishmaniasis in the ELISA-Fe-SODe (prevalence: 53.33%; Table 1 and Fig. 1). Of these eight positive sera, three had antibodies against Fe-SODe from Le. mexicana only and one against Fe-SODe from Le. braziliensis only. The remaining sera tested positive for antibodies from all the parasites studied.

FIG. 1.

Results of the assay of 70 serum samples from dogs in the state of Yucatan (Mexico) by enzyme-linked immunosorbent assay using the Fe-SODe from promastigotes of Leishmania (Leishmania) mexicana (SODe-Lm, ), Leishmania (Viannia) braziliensis (SODe-Lb, ), and epimastigotes of Trypanosoma cruzi (SODeCRU, ) as antigen at a dilution of 1/200. The mean and standard deviation of the optical densities of the control sera were used to calculate the cutoff value (mean + 3 × standard deviation). SODe, superoxide dismutase.

Table 1.

Relationship of Positive Sera of Stray Dogs from the State of Yucatan (Mexico) with Trypanosomatids by Enzyme-Linked Immunosorbent Assay Against Different Antigen Fractions of Leishmania spp. and Trypanosoma cruzi

ELISA ^a (serum titers 1/200)
	H				Fe-SODe
Serum	LM	LB	LPA	CRU	LM	LB	LPA	CRU
Tulum
1					+	+
4					+
5					+
8						+	+	+
10					+
11					+	+		+
13					+	+		+
14						+
Subtotal	0	0	0	0	6	5	1	3

Celestun
17	+				+	+
18					+
21		+			+	+
22		+			+	+		+
25				+		+		+
26					+
30						+	+
31				+	+			+
36					+
38		+			+	+
39					+	+
40					+
41				+				+
42					+	+
46					+	+
48					+
49					+
50					+	+
51					+			+
52				+	+	+		+
53				+	+	+		+
55						+
58				+	+			+
59					+
60					+
61						+
62						+
63					+	+
64					+	+	+	+
66						+
Subtotal	1	3	0	6	23	18	2	9
Total	1	3	0	6	29	23	3	12

Antigen fraction: Total parasite extract (H) and superoxide dismutase excreted by promastigotes from Leishmania mexicana (Fe-SODeLM), Leishmania braziliensis (Fe-SODeLB), and Leishmania panamensis (Fe-SODeLPA) and by epimastigotes from Trypanosoma cruzi (Fe-SODeCRU).

Fe-SODe, excreted iron-superoxide dismutase.

ELISA-H indicated that four of the 55 sera from Celestún (7.27%) were positive for one species of Leishmania (one for Le. mexicana and three for Le. braziliensis), although none of these were positive for more than one Leishmania spp. The ELISA test with Fe-SODe as the antigen fraction identified 29 positive sera (52.72%), 15 of which were positive against at least one Leishmania sp. The remaining 14 tested positive for antibodies from several Leishmania spp.

The use of Fe-SODe as the antigen fraction is highly sensitive for the detection of leishmaniasis, although it nevertheless results in a small but significant number of false-positive results, as is also the case with Chagas' disease (Marín and Sánchez-Moreno 2009). To demonstrate the possible absence of a cross-reaction with other members of the same family, such as T. cruzi, ELISA studies were performed using whole extract and Fe-SODe from T. cruzi as the antigen fractions in combination with the 70 sera collected. Only six sera (10.90%), all from Celestún, tested positive when using whole-parasite extract as the antigen, although none of them tested positive for Leishmania sp. In contrast, 12 sera, 3 from Tulum and 9 from Celestún, tested positive when using Fe-SODe as the antigen fraction. Two of the sera from Tulum tested positive for both Le. mexicana and Le. braziliensis, and the other tested positive for both Le. braziliensis and Le. panamensis. Only one of the sera from Celestún tested positive for T. cruzi only, with the remainder testing positive for T. cruzi and at least one other Leishmania sp. (Table 2).

Table 2.

Prevalence of Positive Sera of 70 Stray Dogs from the State of Yucatan (Mexico) to Trypanosomatids by Enzyme-Linked Immunosorbent Assay and Western Blot Against Different Antigen Fractions of Leishmania sp. and Trypanosoma cruzi

	No. of sera positive (dilution of sera: 1/200)		Prevalence
Antigen fraction ^a	ELISA	Western blot	ELISA	Western blot
H-LM	1	^b	—	—
Fe-SODeLM	29	33	41.4%	47.1%
H-LB	3	^b	4.3%	—
Fe-SODeLB	23	30	32.8%	42.8%
H-CRU	6	^b	8.6%	—
Fe-SODeCRU	12	20	17.1%	29.0%
H-LPAN	0	^b	0.0%	^—
Fe-SODePAN	3	^b	4.3%	^—

Refer the footnote of Table 1.

Not determined.

None of the techniques currently used for blood screening gave 100% sensitivity and specificity, and therefore, public health organizations currently recommend that at least two tests (based on different methodologies) be performed (Marín and Sánchez-Moreno 2009). Following these recommendations, we assayed all 70 sera by western blot using Fe-SODe from the parasites studied as the antigen fraction and found that 33 (47.14%) tested positive for Le. mexicana and 30 (42.85%) for Le. braziliensis. Figure 2 presents the western blot results, which confirm the positivity found with the ELISA-FeSOD. Further, some of the sera that tested negative in the ELISA-FeSOD tested positive by western blot. Thus, four sera (numbers 23, 25, 55, and 68) tested positive for Le. mexicana and seven (numbers 28, 32, 34, 44, 48, 58, and 65) for Le. braziliensis by western blot only. In the case of T. cruzi, eight sera (numbers 4, 21, 32, 39, 44, 46, 55, and 68) tested positive by western blot only, thus confirming that this technique is more sensitive than ELISA (Marín et al. 2009). As very few sera tested positive for Le. panamensis using the Fe-SODe-ELISA technique, none was assayed by western blot. This contrasts with the results obtained with the other parasites (Le. braziliensis, Le. mexicana, and T. cruzi), which confirmed the positivity of the sera and the absence of a cross-reaction (Table 3).

FIG. 2.

Immunoblot of the positive sera from stray dogs from the state of Yucatan (Mexico) at a dilution of 1/200 against SODe from Le. mexicana (A), Le. braziliensis (B), and Trypanosoma cruzi (C). Line M: SODe activity with isoelectric focusing and staining following the technique of Beyer and Fridovich (1987).

Table 3.

Detection of Coinfection in 70 Sera of Stray Dogs of the State of Yucatan (Mexico) by Enzyme-Linked Immunosorbent Assay of Excreted Iron-Superoxide Dismutase

Trypanosomatids ^a	No. of sera positive	Prevalence
LM/LB	9	12.8%
LM/CRU	3	4.3%
LM/LPAN	0	—
LB/CRU	1	1.4%
LB/LPAN	1	1.4%
CRU/LPAN	0	—
LM/LB/CRU	5	7.1%
LM/LB/LPAN	0	—
LB/CRU/LPAN	1	1.4%
LM/LB/CRU/LPAN	1	1.4%

Promastigote forms of Le. mexicana (LM), Le. braziliensis (LB), and Le. panamensis (LPAN) and epimastigote forms of T. cruzi (CRU).

Discussion

In 1993, three dogs infected with leishmaniasis were found to be living with their owners, who also proved to be infected with the disease, in the state of Quintana Roo (in a town 96 km from Tulum, one of the areas of our study) (Velasco Castrejon et al. 2009). This close relationship between dogs and persons infected with leishmaniasis had been noted previously in several countries, including Argentina and Brazil (Taranto et al. 2000, Marco et al. 2005). In general, dogs that are allowed to run loose constitute a considerable risk for their owners, particularly in light of their closeness to humans, by acting as reservoirs for cutaneous leishmaniasis. Indeed, this would explain why sand flies are often found in the dwellings of some country dwellers.

The susceptibility of Mexican dogs to Leishmania spp. is similar to that found in other Latin American countries, although dogs in Mexico appear to be exposed to a higher infection risk than dogs in other countries, thus explaining the high prevalence of this disease in the former (Velasco Castrejon et al. 2009). These findings led us to undertake an epidemiological study of the disease in stray dogs, which are considered to be key reservoirs and to act as the nexus between the wild cycle and the domestic one. The serological evidence obtained from these dogs showed that three species of Leishmania responsible for cutaneous leishmaniasis circulate in Tulum (Quintanta Roo state) and Celestun (Yucatan state). Further, our Fe-SOD–based ELISA and western blot results provide evidence that both Le. braziliensis and Le. mexicana infection occur with unexpectedly high frequency at both locations. Moreover, this is the first time that evidence of Le. panamensis infection has been found in these regions (Andrade et al. 2003, 2005, Velasco Castrejon et al. 2009, Pech-May et al. 2010). These findings are not, however, particularly surprising given the reported presence of Le. braziliensis in neighboring countries such as Belize and Guatemala (Soto et al. 2004, Schnedl et al. 2007) and of Le. panamensis in other countries, such as Nicaragua and Honduras, which, despite being somewhat farther away, are still relatively close (Zeledón et al. 1993).

This is the first report of dogs infected with Le. mexicana in the region of Celestún, which until now was not considered endemic. Further, it has been demonstrated that other species, such as Le. braziliensis and Le. panamensis, are circulating in this region.

The prevalence of Le. mexicana in Tulum is very similar to that found in Celestún (20%), thus indicating that the endemicity of the two zones is similar. Further, vectors involved in the transmission of the pathogen (Lu. cayanensis, Lu. chiapanensis, Lu. olmeca, and Lutzomyia sp.) have been reported in both regions (Ibáñez 1999).

The presence of different antibodies has been demonstrated between the different stocks of Leishmania spp. This finding is not, however, due to a cross-reaction, because the techniques used in this study (ELISA and western blot) indicated the presence of antibodies specific to each of the species used (Marín et al. 2009). Indeed, tests performed with T. cruzi, which is commonly found in stray dogs on the Yucatan Peninsula, confirmed the absence of a cross-reaction with other species of the same family (Ramos-Ligonio et al. 2006). The greater sensitivity of the western blot with respect to ELISA has been demonstrated using Fe-SODe from the parasites as the antigen fraction, although both serological techniques were concordant, with a Kappa index of 0.83.

In summary, the present study suggests that stray and pet dogs may act as reservoirs for leishmaniasis in the Yucatan Peninsula and that at least three Leishmania species (Le. mexicana, Le. braziliensis, and Le. panamensis), the presence of which in this region was previously unknown, are involved. With a high positivity to Le. mexicana and Le. braziliensis (41.4% and 32.8%, respectively), there is also a quite significant positivity of T. cruzi (17.1%). The stray dog can therefore be considered to be a source of transmission of trypanosomiasis, thus suggesting the need for a more in-depth epidemiological study of the dog population, both wild and urban, on the Yucatan Peninsula to design a control plan for these diseases. Such a study could even be extended to cover other Mexican states and neighboring countries. Because of the close relationship between dogs and their owners, a similar study should be performed with the human population of these regions.

Finally, these results again confirm that the FeSOD excreted by the different trypanosomatid species is a good source of antigen for serodiagnosis in epidemiological studies.

Footnotes

Acknowledgments

The authors thank Encarnación Guerrero for technical help in culture media preparation and Ramón Gutiérrez Sánchez for helpful statistical discussion. This work was financially supported by MEC, Spain (CGL2008-03687-E/BOS).

Disclosure Statement

No competing financial interests exist.

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