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Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil

Abstract

INTRODUCTION:

We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil.

METHODS:

Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners’ knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher’s exact tests, followed by logistic regression, was used.

RESULTS:

It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog’s size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023).

CONCLUSIONS:

These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

Keywords:
Risk factors; Leishmania; Dogs; Rural area

INTRODUCTION

Leishmaniases are zoonoses caused by protozoa belonging to the genus Leishmania11. Ross R. (1) note on the bodies recently described by Leishman-Donovan and (2) Further notes on Leishman’s bodies. Br Med J. 1903;2(1):1261-1401.. The species that causes visceral leishmaniasis (VL) in countries in the Americas is Leishmania infantum (syn. L. chagasi)22. Kuhls K, Alam MZ, Cupolillo E, Ferreira GEM, Mauricio IL, Oddone R, et al. Comparative microsatellite of new world Leishmania infantum reveals low heterogeneity among populations and recent old world origin. PLoS Negl Trop Dis. 2011;5(6):e1155..

The main means of transmission of the parasite to dogs and other mammalian hosts is through the bite of females of hematophagous dipterans of the family Psychodidae belonging to the genera Phlebotomus and Lutzomyia, in the Old and New World, respectively, which are infected with promastigote forms of Leishmania spp.33. Deane LM, Deane MP. Leishmaniose visceral urbana (no cão e no homem) em Sobral, Ceará. O Hospital. 1955;47:75-87.,44. Young DG, Duncan MA. Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Memoirs of the American Entomology Institute. Gainesville: Associated Publishers -American Entomological Institute; vol 54. 1994.. The species Lutzomyia (Lutzomyia) longipalpis is considered to be the main transmitter of the parasite in Brazil. This vector species feeds on a wide variety of vertebrate hosts, such as birds, wild and domestic mammals, and humans55. Ministério da Saúde (MS). Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Manual de Vigilância e Controle da Leishmaniose Visceral. 1ª Edição. Brasília: MS; 2014. 122 p..

Although several wild hosts have been identified in urban areas, Canis familiaris is the domestic host, and is considered to be the main reservoir of infection for humans66. Gontijo CMF, Melo MN. Leishmaniose visceral no Brasil: quadro atual, desafios e perspectivas. Rev Bras Epidemiol. 2004;7(3):338-49.. Clinical manifestations of visceral canine leishmaniasis (CanL) are characterized by dermatological symptoms: flaking and excessive depigmentation, which normally develop on the head, and which differ to other parts of the body, with itching, dry skin, hair loss and areas of hyperkeratosis and lignification, and onychocryphosis. They may also present ulcers and small intradermal nodules, digestive symptoms (intestinal hemorrhage), respiratory symptoms (runny nose), eye symptoms (conjunctivitis, blepharitis, corneal enlargement and opacity) and general symptoms (apathy, anorexia, anemia, limb edema, hyporexia, weight loss and lymphadenomegaly)77. Feitosa MM, Ikeda FA, Luvizotto MCR, Perri SHV. Aspectos clínicos de cães com leishmaniose visceral no município de Araçatuba, São Paulo, Brasil. Clín Vet. 2000;5(28):36-44..

Many risk factors (RFs) for the occurrence of VL have been listed, thus indicating possible interactions between the links that make up the epidemiological chain, such as vectors, hosts and the environment88. Dye C. Leishmaniasis epidemiology: the theory catches up. Parasitol. 1992;104:s7-18.,99. Gavgani ASM, Mohite H, Edrissian GH, Mohebali M, Davies CR. Domestic dog ownership in Iran is a risk factor for human infection with Leishmania Infantum. Am J Trop Med Hyg. 2002;67(5):511-15.,1010. Monteiro EM, Silva JCF, Costa RT, Costa DC, Barata RA, Paula EV, et al. Leishmaniose visceral: estudos de flebotomíneos e infecção canina em Montes Claros, Minas Gerais. Rev Soc Bras Med Trop. 2005;38(2):147-152.,1111. Murray HW. Advances in leishmaniasis. Lancet. 2005;366:1561-77.,1212. Rondon FCM, Bevilaqua CML, Franke CR, Barros RS, Oliveira FR, Alcântara AC, et al. Cross-sectional serological study of canine Leishmania infection in Fortaleza, Ceará state, Brazil. Vet Parasitol. 2008;155:24-31.. Thus, knowledge of the distribution of the disease in endemic areas and possible associations between the disease and RFs can help in developing control strategies1313. Frehse MS, Greca Jr H, Ullmann LS, Camossi LG, Machado JG, Langoni H, et al. Surveillance of canine visceral leishmaniasis in a disease-free area. Rev Bras Parasitol Vet. 2010;19(1):64-6.. In this context, domestic dogs play an important role in the maintenance and spread of the disease. For this reason, factors that may be associated with the risk that these animals may become infected need to be well known1414. Dantas TF. Canine leishmaniosis in South America. Parasit Vectors. 2009;2(1):S1.

Some studies conducted over the last decade have identified certain RFs that are associated with VL in urban regions. These include poor housing conditions, especially with a lack of household waste collection and an irregular or absent sewage system1515. Moreno EC, Melo MN, Genaro O, Lambertucci JR, Serufo JC, Andrade ASR, et al. Risk factors for Leishmania chagasi infection in an urban área of Minas Gerais State. Rev Soc Bras Med Trop . 2005;38(6):456-63.; increased population density of phlebotomine sand-flies1515. Moreno EC, Melo MN, Genaro O, Lambertucci JR, Serufo JC, Andrade ASR, et al. Risk factors for Leishmania chagasi infection in an urban área of Minas Gerais State. Rev Soc Bras Med Trop . 2005;38(6):456-63.,1616. França-Silva JC, Barata RA, Costa RT, Monteiro EM, Machado-Coelho GLL, Vieira EP, et al. Importance of Lutzomyia longipalpis in the dynamics of transmission of canine visceral leishmaniasis in the endemic area of Porteirinha municipality, Minas Gerais, Brazil. Vet Parasitol . 2005;131(3-4):213-20.; breeding of birds in cages in the presence of the vector1515. Moreno EC, Melo MN, Genaro O, Lambertucci JR, Serufo JC, Andrade ASR, et al. Risk factors for Leishmania chagasi infection in an urban área of Minas Gerais State. Rev Soc Bras Med Trop . 2005;38(6):456-63.; and presence of other animals in the peridomestic area, particularly opossums1717. Cabrera MA, Paula AA, Camacho LAB, Marzochi MCA, Xavier SC, Silva AVM, et al. Canine visceral leishmaniasis in Barra do Guaratiba, Rio de Janeiro, Brazil: assessment of risk factors. Rev Inst Med Trop S Paulo. 2003;45(2):790-83., chickens and pigs1818. Moreira Jr ED, Souza VMM, Sreenivasan M, Lopes N, Barreto RB, Carvalho LP. Peridomestic risks factors for canine leishmaniasis in urban dwellings: new findings from a prospective study in Brazil. Am J Trop Med Hyg . 2003;69(4):393-97..

Recently, a cross-sectional study carried out in endemic areas of Cuiabá, state of Mato Grosso, showed a CanL seroprevalence of 22.1%. Animals living in rural settings had a 1.9-fold higher risk of been infected than those in an urban environment. Factors relating to the habits of these animals, such as free access to the external environment and a watchdog function, along with the presence of agricultural activity were probably indicators that predicted Leishmania spp. exposure1919. Almeida ABPF, Sousa VRF, Cruz FACS, Dahroug MAA, Figueiredo FB, Madeira MF. Canine visceral leishmaniasis: seroprevalence and risk factors in Cuiabá, Mato Grosso, Brazil. Rev Bras Parasitol Vet . 2012;21(4):359-65..

Paulan et al. 4949. Paulan SC, Silva DT, Lins AGS, Lima FL, Tenório MS, Tasca KI, et al. O conhecimento sobre leishmaniose visceral: suficiente para controle e prevenção? Rev Ciênc Ext. 2016;12(2):47-60. (2012) used geoprocessing techniques in association with satellite imaging to reveal that the estimated prevalence of CanL in Ilha Solteira, state of São Paulo was low to medium-high, ranging from 10% to 14.5%, depending on the neighborhood studied. The areas with the highest density of CanL cases were close to natural vegetation fragments (at a zoo) and near rural settlements, i.e. farther from the city center.

Spada et al. 2121. Spada JCP, Silva DT, Martins KRR, Rodas LAC, Alves ML, Faria GA, et al. Occurrence of Lutzomyia longipalpis (Phlebotominae) and canine visceral leishmaniasis in a rural area of Ilha Solteira, SP, Brazil. Rev Bras Parasitol Vet . 2014;23(4):456-62. (2014) studied the prevalence of Lu. longipalpis and CanL in the “Cinturão Verde” (green belt) area. They visited 12 properties over a 12-month period and collected biological samples from 32 dogs. Once a month, insects were caught using CDC (Centers for Disease Control and Prevention) traps. It was found that the vector was present on 100% of the properties, and that 31.25% of the dogs were positive for CanL.

The "Cinturão Verde" has a considerable human and canine population, which presents suitable conditions for vector maintenance, and is located near the urban perimeter of the city; this area represents an RF for maintenance of local disease and spread of this zoonosis to the urban area, if preventive measures are not implemented.

Thus, the objective of this study was to determine the RFs associated with Leishmania exposure among dogs in the “Cinturão Verde” of Ilha Solteira, SP, Brazil.

METHODS

Study area

This study was conducted in a rural area referred to as the “Cinturão Verde” (Green Belt), which belongs to the municipality of Ilha Solteira (51°06’35” W and 20°38’44” S). The Cinturão Verde occupies an area of ​​880.46 hectares (ha) and is divided into agricultural production areas (563.29 ha); reforestation areas (317.68 ha); talvegues (lines connecting the lowest points of a river bed) (45.65 ha); area used for hydroelectric construction (227.39 ha); and legally enriched reserves (area with native vegetation cover) (44.12 ha). The entire extent of the Cinturão Verde is surrounded by 77 areas of dry land (non-irrigated) and 14 areas of irrigated land that are distributed among approximately 200 families. These families carry out various functional activities, such as growing vegetables and raising small animals, such as poultry and pigs.

Ethics Committee

The present study was approved by the Ethics Committee for Animal Use (CEUA) of the School of Engineering School of Ilha Solteira (part of São Paulo State University, UNESP). It formed part of a research project entitled "Distribution of the Phlebotomine Entomophase (Diptera: Psychodidae) and Canine Visceral Leishmaniasis Area of the “Cinturão Verde” of Ilha Solteira, State of São Paulo". Approval was granted at an ordinary meeting of CEUA held on May 9, 2011, under protocol no. 002/2011/CEUA. Procedures were performed based on current standards for research involving animal use according to the National Council for Animal Experiment Control (CONCEA).

Study design and dog samples

A cross-sectional study onLeishmaniaexposure in dogs was conducted between February 2012 and February 2013. The sample size was established considering a population of 400 dogs (2 dogs/family) in the study area. Thus, the size of the sample, based on an arbitrary random method and with finite population adjustments of less than 200 dogs and a sampling error of 5%, was estimated to be approximately 250 dogs2222. Kish L. Survey Sampling. 1 ed. New York: John Wiley and Sons, Inc.; 1965. 643 p.. To ensure representativeness of the sample size, it was defined that the methodology for collecting the material should not involve any concentration of samples in any single region of the total area, but rather that the collection of material should cover the entire perimeter of the area. With the aid of a map provided by city authorities and a number of local visits to the study area, land areas and ownership were determined. In total, 104 families were visited, and biological material was collected from all dogs belonging to each family, irrespective of the numbers of dogs.

Blood collection

Blood samples from the dogs were taken directly from the cephalic vein or the external jugular vein, using vacuum flasks without anticoagulant, to obtain serum samples. The whole blood was centrifuged at 900 × g for 10 minutes to separate the serum and was then kept at -20 °C until further use.

Clinical Characterization

At the time of blood collection, the animals were examined clinically by means of general physical examination, and classified according to the clinical signs evident for CanL with one or more clinical signs and without clinical signs. Among the findings on physical examination, cachexia, hyperthermia, hyporexia, dermatological changes such as alopecia, ulcerative skin lesions, flaking, crusts, lymphadenomegaly, periocular lesions, uveitis, conjunctivitis, pale mucous membranes, and onychogryphosis were noted.

Detection of anti-Leishmania antibodies

Anti-Leishmania antibodies were assayed using indirect immunofluorescence antibody tests (IFATs) and indirect enzyme-linked immunosorbent assays (ELISAs) as described by Oliveira et al. 2323. Oliveira TMFS, Furuta PI, de Carvalho D, Machado RZ. A study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test. Rev Bras Parasitol Vet . 2008;17(1):7-11. (2008). Positive control serum was obtained from confirmed CanL cases that had been detected using direct methods. For negative controls, serum from healthy dogs was used.

To perform the ELISA test, the soluble antigen of L. infantum was used at a concentration of 5 µg/mL, diluted in 0.05 M sodium carbonate bicarbonate buffer, pH 9.6. An anti-dog conjugate, rabbit anti-dog IgG coupled to alkaline phosphatase (Sigma Chemical Co, San Luis, Missouri, EUA) was diluted 1: 4000 in phosphate buffered saline (PBS), 0.01 M, pH 7.2 with 0.05% Tween-20 (PBS-Tween). As a substrate, paranitrophenylphosphate diluted to 1 mg/mL in diethanolamine buffer, pH 9.8 was used. The plates were read in an ELISA reader (Dynex Technologies, Chantilly, Virgínia, USA) at 405 nm. The cut-off point for the ELISA test corresponded to two and a half times the average value of the mean optical density (OD) of the negative reference sera.

To perform IFATs, antigenic substrate was obtained from L. infantum promastigotes grown in RPMI - 1640 medium, at 25°C. Serial dilutions of each serum were performed commencing with a 1:40 dilution. The conjugate was dog anti-IgG linked to fluorescein isothiocyanate (KPL, Milford, Massachusetts, USA) diluted according to the manufacturer's recommendations. Sera were considered as positive when the parasites exhibited fluorescent color throughout the periphery, with a cut-off point of ≥ 1:40.

Questionnaires

To determine RFs, standardized questionnaires were used. At the time of collection of blood from the dogs, the questionnaire was applied to the owners or caregivers of the animals. The information sought through the questionnaires included the identification and characteristics of the dogs, the environment in which the dogs lived, and the degree of knowledge regarding VL among the owners.

Definition

Positivity for Leishmania exposure among the dogs was defined as positive detection of antibodies by means of IFAT or ELISA.

Geographic location of the animals

The locations were georeferenced using the Global Positioning System (GPS). These data were imported into a geographic information system (GIS) using the QGIS version 2.18.10 software package (Free Software Foundation, Boston, Massachusetts, EUA) with Open Layers plugin, to visualize the spatial distribution of the data. Finally, dots representing data points were projected into an image layer obtained from the Google Earth database (Figure 1).

FIGURE 1:
Geographic distribution of dogs in the rural area known as "Cinturão Verde" in the municipality of Ilha Solteira, state of São Paulo, Brazil, according to their serological condition. Serologically positive dogs are represented by red markings and negative dogs by white markings.

Statistical analysis

The association between potential RFs and Leishmania exposure in dogs was assessed by means of univariate analysis using the chi-squared test, or Fisher’s exact test, when required, and by means of multivariate logistic regression analysis. Odds ratios (OR) and 95% confidence intervals (CI) were calculated, and p values < 0.05 were considered statistically significant. All analyses were performed using R software (The R Foundation, Vienna, Austria), version 2.15.32424. R Core Team. R: a language and enviroment for statistical computing [Internet]. Vienna: R Foundation for Statistical Computing; 2017 [updated 2017 June 2; cited 2012 Jun 2]. Available from: Available from: https://www.r-project.org/ .
https://www.r-project.org/...
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RESULTS

A total of 250 dogs were analyzed. Anti-Leishmania antibodies were found in 79/250 (31.6%) of the dogs using IFAT, and 72/250 (28.8%) using ELISA. In total, 82/250 (32.8%) of the dogs were positive.

According to clinical classifications, 192/250 (76.8%) dogs were classified as without clinical signs, and 58/250 (23.2%) as with clinical signs. Comparing the clinical conditions with the ratio of positive and negative dogs in the serological tests performed, it can be observed that although the high percentage of negative dogs in the group of dogs without clinical signs for both ELISA (150/192; 78.1%) and IFAT (146/192, 76.0%), there were positive dogs without clinical signs. Regarding dogs with clinical signs, some dogs in this group were negative for both tests performed. Positive dogs without clinical signs (21.9% and 23.9%) were less frequent than positive dogs with clinical signs (51.7% and 56.8%) for ELISA and IFAT, respectively (Table 1).

TABLE 1:
Number (N) and percentage (%) of positive and negative dogs by serological methods (indirect ELISA and IFAT), according to classification as with and without clinical signs in the “Cinturão Verde” area, Ilha Solteira, SP, 2014.

The ELISA cutoff point was an OD of 0.263 and, comparing the clinical classifications of the dogs with the average ELISA titers, it was found that positive dogs without clinical signs had an average OD of 0.642, and positive dogs with clinical signs had an average OD of 0.626. Regarding negative dogs, those with no manifestations exhibited an average OD of 0.254, and dogs with clinical signs had an average OD of 0.244.

Regarding the information obtained from the questionnaire, seven variables relating to dog characteristics were analyzed: hometown, use of repellent collar (impregnated with 4% deltamethrin), place where the dog slept (indoors or outdoors), age, size, sex and dog rearing (free or restrained). However, only animal size was significant by univariate analysis (Table 2) and in multivariate analysis (p < 0.05) (Table 3). Only large animals (such as German Shepherd, Rottweiler and similar breeds) were correlated with susceptibility to Leishmania exposure (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003). Among the 82 samples that were positive for antibodies against Leishmania, 39 (44.8%) were in the group of large dogs.

TABLE 2:
Univariate association analysis of variables relating to risk factors associated with Leishmania exposure, based on dogs (N = 250) in the “Cinturão Verde” area. Ilha Solteira, SP, 2014.
TABLE 3:
Results of multivariate analysis on risk factors relating to seropositivity of dogs for Leishmania infection in the “Cinturão Verde” area. Ilha Solteira, SP, 2014.

Among the 250 dogs sampled in this study, there were 145 males and 105 females. There was greater occurrence of Leishmania exposure in the male population (54/82; 65.9%), but this difference was not significant (OR = 1.63; 95% CI = 0.94-2.82; p = 0.079).

Regarding the age of the animals, 200 (80.0%) were adults (more than one year of age), while 50 (20.0%) were less than one year old. Although the positivity rate was higher among adult animals (69/82; 84.2%), i.e. those over one year of age, the difference was not statistically significant (OR = 0.66; 95% CI = 0.33-1.33; p = 0.250). The small number of young dogs observed in this study suggests that there was little rotation or replacement of dogs in the area studied.

Regarding the habits of these animals, there was no significance (OR = 2.07; 95% CI = 0.15-2.64; p = 0.599). However, it was observed that during the night, among the 82 positive animals, two dogs slept indoors and 50 outside in the yard, and also that among these 82 positive dogs, 37 were restrained and 45 were loose on the properties.

Regarding the environment (Table 4), of the 82 animals that were positive for Leishmania exposure, all lived with other animals (60 with poultry, 29 with cats, 27 with pigs, 24 with horses and 13 with cattle). In the present study, the presence of poultry (hens) cohabiting with dogs was shown to be another RF that influenced the presence or maintenance of infected dogs in the rural regions, as shown by univariate (Table 4) and multivariate analyses (Table 3) (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023). It was observed that of the 82 seropositive dogs, 60 lived on properties on which chickens were also kept.

TABLE 4:
Univariate association analysis of variables relating to risk factors associated with Leishmania exposure, based on environmental factors (N = 250 dogs) in the “Cinturão Verde” area. Ilha Solteira, SP, 2014.

It was found that 20.8% of the dogs sampled were on properties that reported dogs having been put down because of Leishmania exposure (OR = 0.53; 95% CI = 0.26-1.10; p = 0.055). This indicated that the disease was present in this rural environment and that greater attention to it needs to be paid by health authorities. None of the properties reported that any of their dogs had been treated for VL. Among the 82 seropositive dogs, 20 lived on properties that did not have any selective garbage collection.

Finally, regarding the owners’ knowledge concerning VL (Table 5), it was found that 27.2% of the dogs were under the ownership of people who did not know about the disease and how it is transmitted (72.8%); or about its severity and lethality, not only in relation to dogs but also to humans (60.8%). This lack of knowledge concerning the disease among rural populations was a RF for the disease by univariate analyses (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049), whereas this factor ceased to be significant after multivariate analysis. However, it should be noted here that a large proportion (72.8%) of the rural population interviewed reported having a lack of knowledge about the role of the insect vector in relation to transmission of leishmaniasis, and regarding dogs being the main domestic reservoir (64.4%). In addition, 49.2% of the interviewees answered that they were totally unaware of the presence of the vector on their properties, thus corroborating the hypothesis that knowledge concerning CanL among the human population is a crucial factor regarding its prevention.

TABLE 5:
Univariate association analysis of variables relating to risk factors associated with seropositivity of dogs for Leishmania exposure and with their owners’ knowledge about the disease (N = 250 dogs) in the “Cinturão Verde” area. Ilha Solteira, SP, 2014.

DISCUSSION

In other studies carried out in Ilha Solteira, the prevalence of CanL in urban areas ranged from 10% to 14%2020. Paulan SC, Silva HR, Lima EAF, Flores EF, Tachibana VM, Kana C, et al. Spatial distribution of Canine Visceral Leishmaniasis in Ilha Solteira, São Paulo Brazil. Eng Agríc. 2012;32(4):765-74.,2525. Pereira VF, Benassi JC, Starke-Buzetti WA., Silva DT, Ferreira HL, Keid LB, et al. Detection of canine visceral leishmaniasis by conjunctival swab PCR. Rev Soc Bras Med Trop. 2016;49(1):104-6., while it was 37.7% rurally 2626. Paulan SC, Lins AGS, Tenório MS, Pena HFJ, Machado RZ, Gennari SM, et al. Seroprevalence rates of antibodies against Leishmania infantum and other protozoan and rickettsial parasites in dogs. Rev Bras Parasit Vet. 2013;22(1):162-6. and 89% at animal shelters of the Association for Animal Protection2727. Silva DT, Starke-Buzetti WA, Alves-Martin MF, Paixão MS, Tenório MS, Lopes MLM. Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis. Rev Bras Parasitol Vet . 2014;23(2):179-86..

Here, there were seropositive dogs with and without clinical signs for both serological tests performed, but with a greater number of positive dogs with clinical manifestations (51.7% and 56.8%) than positive dogs without clinical signs: 21.9% and 23.9% for ELISA and IFAT, respectively. According to other authors, a large majority of dogs without clinical signs are negative in the different tests routinely used for CanL detection2828. Assis J, Queiroz NMGP, Silveira RCV, Nunes CM, Oliveira TMFS, Noronha-Junior ACF, et al. Estudo comparativo dos métodos diagnósticos para leishmaniose visceral em cães oriundos de Ilha Solteira, SP. Rev Bras Parasit Vet . 2010;19(1):17-25.,2929. Queiroz NMGP, Assis J, Oliveira TMFS, Machado RZ, Nunes CM, Satrke-Buzeti WA. Diagnóstico da leishmaniose visceral canina pelas técnicas de imunoistoquímica e PCR em tecidos cutâneos em associação com a RIFI e ELISA-teste. Rev Bras Parasitol Vet . 2010;19(1):34-40.,3030. Quinnell RJ, Carson C, Reithinger R, Garcez LM, Courtenay O. Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis: longitudinal study and meta-analysis. PLoS Negl Trop Dis . 2013;7(1):e1992.. However, dogs without clinical signs but positive for CanL may be infectious for sand-flies in a proportion similar to those with clinical signs, and are equally important in the epidemiological chain of the disease3131. Laurenti MD, Rossi CN, da Matta VL, Tomokane TY, Corbett CE, Secundino NF, et al. Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector. Vet Parasitol 2013;196:296-300.,3232. Manna L, Reale S, Viola E, Vitale F, Manzillo VF, Michele PL, et al. Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol . 2006;142(3-4):271-80..

Some studies have shown that dogs without clinical signs are generally seronegative, or have antibody levels that are difficult to detect in serological tests. On the other hand, dogs with clinical signs generally exhibit high levels of antibodies3333. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol . 2009;165(1-2):1-18.. In the present study, antibodies against Leishmania were detect by ELISA, with a similar mean OD between groups of dogs with and without clinical signs. Portela et al3434. Portela RWD, Soares RP, Passos GP, Laranjeira DF, Barral TD, Sampaio JR, et al. Leishmania infantum-derived lipophosphoglycan as an antigen in the accurate serodiagnosis of canine leishmaniasis. PLoS Negl Trop Dis . 2019;13(9):e0007720.. (2019) highlighted the importance of adequate diagnosis of infected dogs without clinical signs in endemic areas, since these dogs can remain untreated or unattended. Laranjeira et al. 3535. Laranjeira DF, Da Matta VLR, Tomokane TY, Marcondes M, Corbet CEP, Laurenti MD. Serological and infection statuses of dogs from a visceral leishmaniasis-endemic area. Rev. Saude Publ. 2014;48(4):563-71. (2014) point out that infected dogs lacking clinical signs can develop active infections, representing a source of infection for other dogs and humans, and that even after a recent infection, they produce specific antibodies at high levels before developing clinical signs. Likewise, Assis et al. 3636. Assis J, Queiroz NMP, Silveira RCV, Nunes CM, Oliveira TMFS, Noronha J, et al. Estudo comparativo dos métodos diagnósticos para Leishmaniose Visceral em cães oriundos de Ilha Solteira, SP. Rev Bras Parasitol Vet . 2010;19(1):17-25. (2010) identified an animal without clinical signs and showing high titrates on ELISA and IFAT tests, with moderate to intense parasitic grades in the spleen and liver tissues, respectively, by immunohistochemical and histochemical examination.

During an epidemiological evaluation of the southeastern and southern regions of Spain, it was observed that the seroprevalence of CanL gradually increases with the size of the animal. This characteristic was found to be an RF for the disease3737. Galvez R, Miro G, Descalzo MA, Nieto J, Dado D, Martin O, et al. Emerging trends in the seroprevalence of canine leishmaniasis in the Madrid region (central Spain). Vet Parasitol . 2010;169(3-4):327-34., which supports our results. Besides the fact that all dogs are susceptible to Leishmania exposure, Feitosa et. al. 77. Feitosa MM, Ikeda FA, Luvizotto MCR, Perri SHV. Aspectos clínicos de cães com leishmaniose visceral no município de Araçatuba, São Paulo, Brasil. Clín Vet. 2000;5(28):36-44. (2000) also correlated greater frequency of CanL cases with larger dogs. Since these serve as guard dogs, they are kept in peridomestic areas, and thus are probably more exposed to the vector. Penaforte et al. 3838. Penaforte KM, Belo VS, Teixeira-Neto RG, Ribeiro RAN, Oliviera RB, Schettini DA, et al. Leishmania infection in a population of dogs: an epidemiological investigation relating to visceral leishmaniasis control. Rev Bras Parasitol Vet . 2013;22(4):592-6. (2013) also studied this association and suggested that large dogs suffer more sand-fly bites because they are used as guard dogs, living outside houses.

Almeida (2012)1212. Rondon FCM, Bevilaqua CML, Franke CR, Barros RS, Oliveira FR, Alcântara AC, et al. Cross-sectional serological study of canine Leishmania infection in Fortaleza, Ceará state, Brazil. Vet Parasitol. 2008;155:24-31. and Figueiredo et. al. 3939. Figueiredo MJFM, Souza NF, Figueiredo HF, Meneses AMC, Silva-Filho E, Nascimento GG. Fatores de risco e classificação clínica associados à soropositividade para leishmaniose visceral canina. Rev Bras Saúde Prod Anim. 2014;15(1):102-6. (2014) also found no gender-based predisposition. However, Medeiros et al. 4040. Medeiros CFO, Melo AGC, Lima AKF, Silva ING, Oliveira LC, Silva MC. Perfil hematológico de cães com leishmaniose visceral no município de Fortaleza, Ceará. Ciênc Anim. 2008;18(1):33-40. (2008) observed greater predisposition among male dogs, while Amóra et al. (2006) found a higher percentage of Leishmania infections among bitches in rural areas.

Contrary to our research, Figueiredo et al. 3939. Figueiredo MJFM, Souza NF, Figueiredo HF, Meneses AMC, Silva-Filho E, Nascimento GG. Fatores de risco e classificação clínica associados à soropositividade para leishmaniose visceral canina. Rev Bras Saúde Prod Anim. 2014;15(1):102-6. (2014) found higher positivity among young dogs. Similarly, Moreno; Alvar4242. Moreno J, Alvar J. Canine leishmaniasis: epidemiological risk and the experimental model. Trends Parasitol. 2002;18(9):399-405. (2002) found that dogs younger than three years of age and older than seven were at higher risk of contracting CanL, and that the first of these groups was more susceptible than the second.

Amorá et al.4141. Amóra SSA, Santos MJP, Alves ND, Costa SCG, Calabrese KS, Monteiro AJ, et al. Fatores relacionados com a positividade de cães para leishmaniose visceral em área endêmica do Estado do Rio Grande do Norte, Brasil. Ciênc Rural. 2006;36(6):1854-9. (2006) observed that the greatest number of seropositive dogs had semi-domestic habits, which they explained by suggesting that these animals are more exposed to vector action. Some research has shown that dogs from endemic areas exposed at night can be stung by hundreds of sand-flies. This continuous exposure may favor seroconversion and development of the disease, since the parasite is continuously introduced into the skin of these animals4343. Solano-Gallego L, Koutinas A, Miro G, Cardoso L, Pennisi MG, Ferrer L, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol . 2009;165(1-2):1-18..

Although other vertebrate animals can serve as food sources for sand-flies, and favor their maintenance in areas close to homes, some, such as chickens, can also reduce the number of infectious bites in dogs4444. Silva RBS, Porto ML, Barbosa WO, Souza HC, Marques NFSP, Azevedo SS, et al. Seroprevalence and risk factors associated with canine visceral leishmaniasis in the State of Paraíba, Brazil. Rev Soc Bras Med Trop. 2018;51(5):683-8.. However, Azevedo et al. 4545. Azevedo MAA, Dias AKK, Paula HB, Perri SHV, Nunes CM. Avaliação da leishmaniose visceral canina em Poxoréo, Estado do Mato Grosso, Brasil. Rev Bras Parasitol Vet . 2008;17(3):123-7. (2008), verified that association of the prevalence of seropositivity in dogs is related to cohabitation with other species: chickens were the most frequent cohabitees among the positive dogs, followed by pigs and horses. Borges et al. 4646. Borges BKA, Silva JAS, Haddad JPA, Moreira EC, Magalhãaes DF, Ribeiro LML, et al. Presença de animais associada ao risco de transmissão da leishmaniose visceral em humanos em Belo Horizonte, Minas Gerais. Arq Bras Med Vet Zootec. 2009;61(5):1035-43. (2009) demonstrated that poultry have immense potential for attracting sand-flies, and noted that chickens deserve special attention because of their higher frequency among households, as well as their potential for generating a favorable environment for procreation of sand-flies because of the organic waste that they produce. In another study, Barboza et al. 4747. Barboza DCPM, Leal DC, Souza BMPS, Carneiro AJB, Gomes Neto CMB, Alcânatara ACD, et al. Inquérito epidemiológico da leishmaniose visceral canina em três distritos sanitários do Município de Salvador, Bahia, Brasil. Rev Bras Saúde Prod Anim . 2009;10(2):434-47. (2009) observed the co-presence of chickens, pigs and horses, but found that cats were the most frequent cohabitees with seropositive dogs.

Recently, a cross-sectional study conducted in endemic areas of Cuiabá (MT) has shown that dogs living in rural settings were 1.9 times more likely to acquire the infection than were those in urban environments1919. Almeida ABPF, Sousa VRF, Cruz FACS, Dahroug MAA, Figueiredo FB, Madeira MF. Canine visceral leishmaniasis: seroprevalence and risk factors in Cuiabá, Mato Grosso, Brazil. Rev Bras Parasitol Vet . 2012;21(4):359-65.. Factors relating to the dogs’ habits, such as free access to the streets and serving a guard function, as well as the presence of agricultural activity, were considered to be indicators that predicted infection by Leishmania spp. In addition, Costa et al. 4848. Costa CHN, Werneck GL, Rodrigues Jr. L, Santos MV, Araújo IB, Moura LS, et al. Household structure and urban services: neglected targets in the control of visceral leishmaniasis. Ann Trop Med Parasitol. 2005;99(3):229-36. (2005) and Moreno et al. 1515. Moreno EC, Melo MN, Genaro O, Lambertucci JR, Serufo JC, Andrade ASR, et al. Risk factors for Leishmania chagasi infection in an urban área of Minas Gerais State. Rev Soc Bras Med Trop . 2005;38(6):456-63. (2005) observed that poor housing conditions, open sewage ditches, lack of household waste collection, and irregular or absent disposal of sewage were RFs for Leishmania infection in urban areas.

Moreno et al. 1515. Moreno EC, Melo MN, Genaro O, Lambertucci JR, Serufo JC, Andrade ASR, et al. Risk factors for Leishmania chagasi infection in an urban área of Minas Gerais State. Rev Soc Bras Med Trop . 2005;38(6):456-63. (2005) reported in a study conducted in the metropolitan region of Belo Horizonte that the likelihood that a population would be affected by CanL was six times higher for people who did not know about the vector than for those who were aware of it.

Our data have reinforced the hypothesis that many people still have poor knowledge regarding leishmaniasis and how it is transmitted. This corroborates a recent report by Paulan et al. 4949. Paulan SC, Silva DT, Lins AGS, Lima FL, Tenório MS, Tasca KI, et al. O conhecimento sobre leishmaniose visceral: suficiente para controle e prevenção? Rev Ciênc Ext. 2016;12(2):47-60. (2016), who found that rural families established in the "Estrela da Ilha" rural settlement in Ilha Solteira, SP, presented fragmented knowledge concerning the disease, thus resulting in inefficient practices of prophylactic measures against leishmaniasis among humans and dogs in this rural area.

Changes in attitudes in populations is a goal to be achieved over time, since this involves cultural changes, which seem to be a crucial factor regarding the difficulty in attaining control over this zoonosis. According to Borges et al. 5050. Borges BKA, Silva JAS, Haddad JPA, Moreira EC, Magalhãaes DF, Ribeiro LML, et al. Avaliação do nível de conhecimento e de atitudes preventivas da população sobre a leishmaniose visceral em Belo Horizonte, Minas Gerais, Brasil. Cad de Saúde Pública. 2008;24(4):777-84. (2008), knowledge of the forms of VL transmission and vector recognition decreases the risk of contracting leishmaniasis by a factor of 0.79, while lack of knowledge about the disease increases the risk by a factor of 2.57.

CONCLUSIONS

The RFs associated with occurrence of Leishmania exposure in domestic dogs on properties of the “Cinturão Verde” in Ilha Solteira, SP, were large body size among the dogs, the presence of chickens, and lack of knowledge regarding Leishmania among dog owners. After adjustment through multivariate analysis, only dog size and the presence of chickens were related to the presence of Leishmania exposure among the dogs. However, we must emphasize that this probably happened due to the fact that most of the local population had no knowledge about the disease.

ACKNOWLEDGEMENTS

We thank the Foundation for Research Support of the State of São Paulo (FAPESP) for grants (Nº. 2012/12066-3 and Nº. 2011/07580-7) and the "Cinturão Verde" families who kindly assisted during the CanL surveys.

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  • Financial Support: This work was supported by grants from São Paulo Research Foundation (FAPESP; n. Nº. 2012/12066-3 and Nº. 2011/07580-7) and from the Coordination Office for Improvement of Higher Education Personnel, Brazil (CAPES).

Publication Dates

  • Publication in this collection
    11 Sept 2020
  • Date of issue
    2020

History

  • Received
    19 Feb 2020
  • Accepted
    19 May 2020
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