Spatial Distribution of Leishmania spp. in Dogs of Urban Area of Araçatuba, São Paulo, Brazil

1. Introduction

Visceral leishmaniasis (VL) is a zoonotic disease caused by the protozoan Leishmania infantum (syn. Leishmania chagasi) in Brazil [1]. It is transmitted by female sandflies [2], Lutzomyia longipalpis [3], Lu. Cruzi [4] and possibly by Pintomyia fischeri, which was incriminated as a potential vector in the State of São Paulo [5]. Approximately 50,000 to 90,000 new cases of human visceral leishmaniasis (HVL) are estimated annually, with Brazil being one of the ten countries with the highest prevalence of this disease [6].

In the state of São Paulo, Lu. longipalpis was detected for the first time in the city of Araçatuba, SP, in 1997 [7]. In the same municipality, in 1998 and 1999, autochthonous cases of canine visceral leishmaniasis (CVL) [8] and human visceral leishmaniasis [9] were reported, respectively.

Since then, there has been an expansion of HVL in the State of São Paulo, with autochthonous human cases reported in 18,45% (119/645) of municipalities by 2022 [10]. The emergence and spread of this zoonosis are associated with anthropogenic and climatic behaviors, migrating from the northwest to the southeast of the state [11]. In the last update from the Ministry of Health, on August 20, 2024, the municipality of Araçatuba had 229 human cases with 23 deaths [12].

Domestic dogs are seen as an important reservoir of L. infantum in Brazil [13]. It is important to note that there are reports that the presence of the vector increases the prevalence of canine disease and increases the possibility of human infection [9,14]. For this reason, dogs are the target of a control program in some countries, such as Brazil [15]. In 2018, 574 cases of CVL were registered in the municipality of Araçatuba (data from the Municipal Center for Zoonosis Control of Araçatuba, SP, unpublished), through the canine survey, however it was not carried out in the entire municipality.

As of Decree No. 51,838, of March 14, 1963, the euthanasia law for seropositive dogs became national, as well as the diagnosis of CVL as a prevention and control measure [16].

As of 2012, the Ministry of Health (MS) recommended the protocol with the use of the Dual Path Platform (DPP), as a screening test, and the Enzyme-Linked Immunosorbent Assay (ELISA) as a confirmatory test [17].

The epidemiological framework of this parasitosis involves the vector and the wild and domestic reservoirs and humans, all with different dispersion and movement capacities, therefore, the processes involved in this infection are spatially dependent [18]. Considering that HVL and CVL have a marked impact on Public Health, this study was carried out with the aim of investigating the spatial distribution of dogs infected by Leishmania spp. through DPP and ELISA serological tests, as well as analyzing the vector density in the urban region of Araçatuba, SP, Brazil.

2. Materials and Methods

2.1. Period and Study Area

The study was developed during the month of March 2018, in the urban area of the municipality of Araçatuba, located in the northwest region of the state of São Paulo, Brazil, with an area of 1,167.129 km2 and an estimated population of 195,874 inhabitants [19].

The city was divided into eight areas, with five sectors each, except for the eighth area that had only one sector, totaling 36 sectors, according to the Division of the Dengue Surveillance and Control Program (DPVCD). In each sector, two houses were selected with a minimum distance of 250 m between them, obeying the dispersion in the urban area of the species Lu. longipalpis [20].

2.2. Residences Selection

The 72 houses were selected by convenience sampling, in blocks where cases of human or canine visceral leishmaniasis had been confirmed in the last four years, based on information shared by the Superintendence of Endemic Control of Araçatuba, SP - SR-09 (SUCEN). As an exclusion criterion, residences where the owners stated that they had vaccinated their dogs against visceral leishmaniasis were not included. All houses were georeferenced for later spatial analyses.

2.3. Experimental Design

2.3.1. Sample Calculation

The minimum sample size required to execute this project, at a confidence level of 95%, with absolute precision of 10% and considering the canine population of Araçatuba, which was estimated at 39,175 animals [21], was calculated in 96 samples, using a disease prevalence of 50% [22].

2.3.2. Study Population

To ensure safety, a total of 131 domestic dogs were serologically examined, 49 males and 82 females of mixed breed (SRD), living in the 72 selected houses. Regarding age groups, these animals were classified as young, adult and elderly, respectively dogs under two years old, from two to seven years old and over seven years [23].

2.3.3. Sample Collection

Individual blood samples of 3.0 mL were collected by cephalic venipuncture with disposable syringes and needles and stored in tubes containing Ethylenediamine Tetraacetic Acid (EDTA). Blood serum from the samples were obtained by centrifugation at 1000 g for 10 minutes and stored at -70°C until the time of serological analysis.

2.3.4. Phlebotomine Collections

Phlebotomine collections were performed using CDC light traps (Figure 1), which were placed in the houses of all project participants. Two traps were installed, one inside and the other outside the residence. Thus, the captures of these insects were conducted over three consecutive nights, with an exposure period from 7:00 p.m. to 7:00 a.m. At the end of this period, the collection bags were removed and placed in plastic bags and taken to the laboratory.

The captured sandflies were analyzed under a stereomicroscope to separate them from the other insects (Figure 2).

2.3.5. Investigation and Environmental Observation Form

The investigation form (Appendix A) for each dog was filled out by its tutor, to obtain specific data, such as gender, age range and lifestyle - domesticated animals (those dependent on their owners who leave the home accompanied and restrained by collars) or semi-domiciled animals (dependent on their owners, who remain outside the home for an indefinite period).

The questionnaire (Appendix B) was carried out as an individual interview, being applied to obtain data from each participating resident, with the aim of defining the epidemiological variables that could be correlated with the occurrence of VL, as well as the presence of other domestic animals, vegetation and fruit trees.

2.4. Laboratory Tests

Two serological tests were used to detect antibodies to Leishmania spp. in dogs, the first being the rapid immunochromatographic test, Dual-Path Platform (TR DPP^® - Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil) used with recombinant rK39 proteins from L. chagasi and Protein A Conjugated to Colloidal Gold [24]. The confirmatory test for samples positive for DPP was the Enzyme-Linked Immunosorbent Assay (ELISA - Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil) to detect Anti-L. major-like antibodies. The cut-off was defined based on the manufacturer’s instructions and considered from the average optical density of the negative controls multiplied by two [25]. Both tests were performed according to the guidelines recommended by the manufacturers.

2.5. Statistics

Data analysis consisted of descriptive statistics and multivariate logistic regression analysis to verify risk factors for CVL. Statistics were considered significant when p<0.05.

2.5.1. Kernel Analysis

This analysis allows visualization of both the spatial distribution and the intensity of events [26]. A quartic Kernel intensity function was used to compose a smoothed surface, the value of which is proportional to the intensity of events per unit area [27]. The analysis was estimated for both seronegative and seropositive animals, using the QGIS VERSION 3.2 software, and all maps were created using it.

2.5.2. Local Clustering

The spatial scanning method was performed using the SatScan^® software version 9.5. Thus, cases (seropositive) and controls (seronegative) were considered for analysis using the statistical model with Bernoulli distribution. For each potential cluster, the likelihood ratio test was calculated comparing the hypothesis that the risk of the disease is greater inside the circle against the hypothesis that the risk is equal for the areas inside and outside the circle. The circle with the maximum likelihood ratio value is considered the most likely cluster [26]. The significance level considered for the clusters was p<0.05.

2.5.3. Vector Density

The vector density in each of the eight study areas was estimated by calculating the number of vectors captured by their geographic area (in km²).

2.6. Ethics Committee

The study was approved by the Animal Use Ethics Committee (CEUA) of the Araçatuba School of Dentistry and the Araçatuba School of Veterinary Medicine (FMVA) UNESP, Araçatuba Campus, FOA process No. 00870-2016 (Appendix C) and by the Research Ethics Committee (CEP) of the Araçatuba School of Dentistry-UNESP, Araçatuba Campus, FOA process No. 61241216.4.0000.5420 (Appendix D).

3. Results

From the analysis of cases and controls, it was observed that dogs seropositive for Leishmania spp. were concentrated mainly in the northern region (Figure 3). Seronegative dogs were found throughout the region, with greater evidence in the center and south of the study area (Figure 3). The density of seronegative dogs was much higher than that of seropositive dogs. However, although these are different areas, no significance was found in the spatial prevalence (p>0.05) throughout the study region, according to the local cluster analysis. There were two residences with seropositive and negative dogs.

Of a total of 131 dogs, 27 (20.61%; 95% CI: 14.57-28.33%) were reactive in the DPP test for Leishmania spp. and 11 of these (40.74%; 95% CI: 24.51-59.27%) were confirmed in the ELISA test, resulting in a prevalence of 8.4% (11/131).

The descriptive and regression statistics analysis showed no association (P < 0.05) between the variables analyzed and the occurrence of Leishmania spp. in the dogs evaluated (Table 1).

Lu. longipalpis was the only insect vector of L. infantum captured in the city of Araçatuba, SP, during the study period. A total of 120 specimens of the vector were captured in seven of the eight areas investigated. This may have been evidenced by the fact that area eight had only two houses and the other areas had ten.

Regarding the distribution of Lu. longipalpis, it was observed that there was no correlation between the areas of greater vector density and higher rates of occurrence of CVL (Figure 4). Thus, areas one (A1) and two (A2) demonstrated the same vector density. However, in the first area, a higher prevalence of seropositive dogs for Leishmania spp. was observed, 36.36% (4/11) compared to the second area, 18.18% (2/11). The fourth area (A4) presented a low prevalence, 9.09% (1/11), but a higher vector density.

Areas three (A3) and six (A6) showed the same vector density, however, the prevalence of seropositive animals was 18.18% (2/11) and 9.09% (1/11), respectively. In areas five (A5) and seven (A7) the same density of insects was also observed, however the prevalence of seropositive dogs was 9.09% (1/11) and 0% (0/11), respectively.

4. Discussion

For the first time, the spatial distribution and analysis of seropositive and seronegative canine cases by DPP and ELISA tests for Leishmania spp. is represented in the urban area of the municipality of Araçatuba, SP. A previous study was conducted with other serological tests [9].

Although there was no significant difference in prevalence in the different areas, the animals seropositive for Leishmania spp. were concentrated in the northern region, with some positive cases in the southern region. In another study, there was also a higher prevalence in the northern region, with no seropositivity in the southern region [9], which demonstrated that there was probably dispersion in this urban area of the municipality over time.

The seroprevalence of CVL in the study was 8.4%, consistent with studies carried out in the municipality, such as 12.1% in the canine serological census [9] and 8.1% in a specific region of the city [18].

In this study, 131 dogs were examined, of which 27 were seropositive for Leishmania spp. by DPP and only 11 were confirmed by ELISA. This difference can be explained by the fact that the rapid test presents a sensitivity and specificity based on rK39 proteins, and studies have already demonstrated a greater sensitivity for detecting symptomatic than asymptomatic dogs [28,29,30].

In endemic cities, such as Araçatuba, where euthanasia of dogs seropositive for CVL is adopted as a control measure, it was found that the population tends to be younger due to the replacement of dogs by their owners, and, therefore, more susceptible to infectious diseases [23,31]. On the other hand, in areas where euthanasia is not frequently applied, the prevalence of CVL tends to increase with age [32]. Although this research evaluated an area with frequent euthanasia and replacement of dogs, the results did not observe a significant difference in infection between age groups.

In the present study, although there were more infected males than females, this result was not significant. This has been previously observed as well as observed by other authors [32]. However, it has been noted that Leishmania spp. infection in male dogs was more prevalent than in females [33]. The lifestyle of dogs kept in the peridomicile during the night, period of greatest activity of Lu. longipalpis [34], may increase the risk of exposure to phlebotomine sandflies and consequently to this parasitosis [35]. The vector is active from twilight onwards [2]. Thus, this may possibly be a matter of environmental and behavioral exposure and not due to gender [36]. Or even a style of raising, in which owners keep their animals indoors or outdoors. In the present study, although 100% of the animals that sleep indoors were seronegative, this variable was not significant.

Of the specimens captured, Lu. longipalpis was the only vector found in seven of the eight areas investigated. The low vector density in area eight can be explained by the number of residences in this area and, consequently, the absence of LVC.

Although the entomological collections were carried out in only one month, it was noted that these insects expanded throughout the urban area of Araçatuba, SP, as in a previous study, they had a higher density in the central and southern regions of the city [9].

Regardless of no association was found between the occurrence of antibodies against Leishmania spp. and the presence of chickens, this can probably be explained by the low frequency of households with poultry.

Chickens can be a source of shelter and food for sand flies, as well as vegetation in the home [37], especially fruit trees [38], which have already been identified as risk factors for the presence of this disease due to attracting sand flies [39]. However, in the present study this factor also did not demonstrate significance for canine cases. The central region (A4) continues to be an area of greater vector density, as highlighted in another study [9]. This can be explained by the fact that these areas have residences with large backyards, with medium to large trees, mostly fruit trees, causing shade and humidity in the soil, as well as the raising of other animals such as cats, birds and chickens, as we know these factors can favor the breeding and proliferation of sand flies.

In the present study, no association was observed between the distribution of the vector and the residence of dogs infected by Leishmania spp. This result has already been observed [9] and may be due to the fact that vector captures were made at a specific time, while the serological result for Leishmania spp. may be due to recent or late infections, since high antibody titers in dogs can remain for more than a year [40,41].

5. Conclusions

The spatial analysis using the tests recommended by the Ministry of Health for the diagnosis of canine visceral leishmaniasis was performed for the first time in the urban region of Araçatuba, SP.