Diagnosis of Leishmania ( Leishmania ) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas , State of Tocantins , Brazil Identificação de cães infectados por Leishmania ( Leishmania ) chagasi e sua relação com aspectos ambientais e san

Introduction: The aim of the present study was to identify the presence of Leishmania (Leishmania) chagasi infection in dogs in the City of Palmas, Tocantins, Brazil, using the PCR technique to list the hot spots of infected dogs in the city and associate their occurrence to significant environmental changes at capture sites. Methods: DNA was extracted from blood of dogs, and the PCR were performed with primers RV1/RV2. After screening the population studied, the regions of the city that had the highest occurrence of canine infection were detected. These sites were visited, and ecological parameters denoting anthropogenic disturbance were evaluated. Results: Some important features were listed in the regions visited, such as low urbanization, lack of public collection of sewage, limited garbage collection, vacant lots with tall vegetation, decaying organic matter, and, most importantly, the occurrence of stray dogs and poultry in homes. Conclusions: The methodology for screening the population was very efficient, especially in evaluating a large number of individuals in a short time, with a high degree of automation. The results indicate an association between the observed parameters and the occurrence of infection in dogs. The model presented in the city is ideal for studies of disease progression and expansion and for the evaluation of control measures adopted for canine VL.

In Latin America, visceral leishmaniasis (VL) is caused by Leishmania (Leishmania) chagasi (Cunha & Chagas, 1937), a kinetoplastid protozoan member of the Leishmania donovani complex.The disease is transmitted mainly by the bite of the phlebotomine Lutzomyia longipalpis.VL is a consumptive debilitating disease whose clinical manifestations are intrinsically dependent on the type of immune response expressed by the infected animal 1,2 .Dogs are considered the main reservoir of the disease in urban environments, which have afforded these animals a central role in this disease's transmission cycle 3,4 .
In Brazil, VL is considered an endemic disease, though outbreaks occur more or less often due to poor immune response of hosts 4 and to an ecoepidemiological scenario that favors the proliferation of infected vector populations 5 .In 2003, the State of Tocantins, Northern Brazil, recorded the highest prevalence of leishmaniasis in the country, with 20 cases per 100,000 inhabitants 6,7 .In 2008 and 2009, the recorded prevalence was 36.8 and 33 cases per 100,000 inhabitants, respectively.In 2010, 21 municipalities in Tocantins were considered a priority region in efforts of VL surveillance and control (Secretaria de Saúde do Estado do Tocantins -SESAU/TO: unpublished data).
The diagnosis of leishmaniasis poses one of the most significant problems concerning the disease, which in most cases may render ineffective VL surveillance and control measures.Despite the difficulties associated to the interpretation of seroprevalence data, the Health Ministry of Brazil recommends that serological diagnosis be performed based on sampling and population surveys 1 .Also, non-specific cross-reactions and delay between infection and seroconversion occur often 8 .

METHODS
Bigeli JG et al -Relationship between canine leishmaniasis and sanitation aspects Therefore, several polymerase chain reaction (PCR) protocols have been developed to detect Leishmania.The technique has been consistently validated as the fastest and most sensitive and specific one as compared to other diagnosis protocols -apart from its suitability when used in leishmaniasis surveillance programs 2,[8][9][10][11] .Concerning molecular diagnosis, kinetoplast DNA minicircles (KDNA) are good targets in the Leishmania genome, as these present up to 10,000 copies per cell, which increases sensitivity of detection procedures 2,12 .
The present study identifies the presence of L. (L.) chagasi in dogs living in the City of Palmas, State of Tocantins, Brazil, using the PCR protocol.The investigation surveyed hot spots of infected dogs in the city to establish a link between the canine infection by L. (L.) chagasi and marked environmental changes in capture sites.

Study area
Collections were carried out in the City of Palmas (10º12'46"S; 48º21'37"W), located in central State of Tocantins, Brazil.The municipality area was divided in 8 zones according to geographic position and urbanization level to allow the pooling of samples and respective results (Figure 1).

RESULTS
the city's inhabitants, between July 2007 and January 2009.DNA extraction was carried out using 3-to 5-mL blood samples collected from these dogs using sterile tubes containing EDTA 27mM as anticoagulant.

Primer-specific PCR
The PCR was conducted using the pair of primers RV1 (5' -CTT TTC TGG TCC CGC GGG TAG G -3') and RV2 (5' -CCA CCT GGG CTA TTT TAC ACC A -3') 14,15 to detect the 145-bp target sequence in the LT1 fragment, located in the kinetoplast DNA minicircle of the L. donovani complex 2,16 .The PCR were conducted in 1.5U/μL Taq DNA polymerase (LGC™ Biotecnologia), Taq reaction buffer (Tris-HCl 100mM pH 8.5 and KCl 500mM), MgCl 2 1.5mM, deoxyribonucleotide triphosphate (dNTP) 0.2mM, 10pmol each primer, and 300ng DNA of each individual and complemented to a final 20μL volume with water.The reactions were carried out in a thermal cycler (PxE0.2,Thermo Electron Corporation™, Milford, MA, USA) according to the following steps: 94ºC for 5min, 35 denaturation cycles at 94ºC, primer hybridization at 58ºC, extension at 72ºC, 72ºC for 10min, and 4ºC for 10min.All reactions were conducted in triplicate, and the DNA of a L. (L.) chagasi culture (Merivaldo strain, IOC-LC2455, isolated from a VL patient in an endemic area in Jequié) 17 was used as positive control.Finally, the amplicons were analyzed by horizontal electrophoresis on agarose gels 2% (m/v) stained with ethidium bromide 0.2μg/mL at 90V for 90min.Gels were photographed using a video documentation system (Vilber Lourmat™, France).Amplicon visualization was conducted in triplicate, and individuals were assigned a PCR positive or negative status.The molecular analysis results obtained did not influence CCZ standard conduct, as the seropositive animals, evaluated in compliance with the standard methodology defined by the Health Ministry of Brazil and conducted in a laboratory certified by the health authorities, were euthanized in accordance with the Health Ministry guidelines.

Statistical analysis
The statistical analysis of data was conducted using the software BioEstat version 4.0.The Chi-square test was employed to observe the occurrence of statistically significant differences between subpopulations, as defined according to the zoning of the city area, as to the occurrence of canine infection by L. (L.) chagasi.Significance level was 5%.

In loco visits: identification of hot spots
After the PCR analyses were finished, results were analyzed to identify the areas presenting the highest prevalence of dogs infected with L. (L.) chagasi in Palmas, Tocantins.Then, in loco visits were undertaken to determine anthropogenic parameters of ecological disturbance.The parameters assessed were: urbanization level, type of construction, garbage and sewage collection systems, vacant lots, vegetation, presence of dogs and breeding practices, and presence of hens and other animals.The geographic data about the sites were collected using a GPS eTrex H device (Garmin™, Chicago, IL, USA).The parameters observed were written down on spreadsheets and then photodocumented using a digital camera.

Ethical considerations
All experimental procedures were approved by the Project and Research Assessment Committee, Palmas Health Authority, protocol no.52-03/19.

Polymerase chain reaction
The analysis of the 204 samples collected using the PCR protocol indicated the occurrence of 121 (59.3%) of dogs positive for L. (L.) chagasi (Figure 2).

Tocantins region
The data on canine infection prevalence and geographical distribution across the different zones of Palmas are shown in Table 1.The Chi-square test revealed statistically significant difference at 5% level of significance (Chi-square = 52.4;p < 0.0001), indicating that there are zones with comparatively higher prevalence values of canine infection by L. (L.) chagasi in the city of Palmas, Tocantins.Henhouses c + + + a Percentage of the area covered by the public garbage collection system; b percentage of privately owned homes.The regions evaluated did not present sewage collection system; c +: represents presence of the aspect.

Anthropogenic parameters of ecological disturbance
After the zones with the highest prevalence of L. (L.) chagasi infection were established for the population sampled, in loco visits were undertaken in the blocks corresponding to the regions designated as R-1, R-2, and R-5.Among the main aspects observed during the visits were (Table 2): I) low urbanization level, with, on average, 50% of blocks presenting streets paved with asphalt; II) absence of a public sewage collection system, with some homes having their own cesspools (around 80%) and many still discharging sewage into open-air ditches; III) poor public garbage collection that fails to cover all the area, including the zones studied (only 75% of the area); IV) large number of vacant lots presenting tall vegetation, which are used by the population as a landfill for demolition debris, domestic garbage, and organic waste (a very common characteristic in the zones evaluated); V) high number of stray dogs on the streets; and VI) considerable number of homes where dwellers raised poultry, another aspect commonly observed in the geographic evaluation.
The inclusion of PCR in surveillance and control measures against leishmaniasis is a tool that may afford faster and more efficient answers in the fight against the propagation of the disease, especially when specifically applied to evaluate asymptomatic reservoirs (data not shown) and also in scenarios of hosts with poor immune response.The adoption of the PCR protocol in these measures is a practical reality, as the PCR technique affords to detect the causal parasite of VL, independently from the quality of the immune response produced by the infected organism.
PCR is a fast-response technique, as it affords to analyze numerous individuals at once, apart from being subject to little interference from the operator due to its high automation.Mohebali et al. 20 state that the detection of L. (L.) chagasi in samples of infected organs is the gold-standard method to diagnose canine infection by the parasite.However, these samples are obtained using invasive procedures, like aspiration of bone marrow, lymph nodes, and splenic puncture.Serum tests are not appropriate for immunocompromised patients, apart from the fact that false-negative results may occur.PCR has been proven to be as appropriate, or even more so, as the diagnostic methods mentioned above, with the added benefit of producing more timely results.Falah et al. 21and Chargui et al. 22 conducted a study in Kairouan, Tunisia, and observed that PCR was more efficient than immunofluorescence and in vitro culture.Other studies also suggest that PCR offers more potential for a direct, efficient, sensitive, and species-specific diagnosis 8,20 .
As pointed out by Fallah et al. 21, apart from being less invasive, collection of blood samples affords good reproducibility and is better accepted by dog owners, as compared to aspiration puncture of bone marrow, spleen, or lymph nodes.As L. (L.) chagasi are intracellular parasites that infect cells of the mononuclear phagocytic system, like monocytes and macrophages, the use of the leukocyte layer instead of whole blood may increase sensitivity, reducing the interference of potential reaction inhibitors.PCR has gradually become the technique indicated to diagnose leishmaniasis, as conventional parasitology methods are not sufficiently sensitive 23,24 .
In accordance with other papers, peripheral blood samples were analyzed by PCR in the present study, producing particularly motivating results and confirming its use in routine diagnosis of canine VL 21,25 .
The results obtained using PCR suggest that dogs infected with L. (L.) chagasi are widely distributed in the City of Palmas, State of Tocantins, Brazil (Table 1).High prevalence is observed in all regions of the municipality, varying between 39.5% in region R-6 and 75% in regions R-1 and R-2.These data characterize the region as an important endemic area in the country.This endemic status is explained by recent urbanization process the region has undergone in the past 20 years, in a transformation triggered by intense anthropic activities in the period.This characteristic underscores the importance of the City of Palmas in studies on VL progression and on the efficiency of control strategies against the expansion of leishmaniasis.
Here, it was also possible to observe the association between the high prevalence of L. (L.) chagasi in dogs in the area studied and poultry rearing in the homes located therein.These observations are an important finding to take into account, as the presence of a dog in the domestic environment and in the property as a whole does not represent a primordial risk factor in terms of VL infection, as opposed to the presence of a vector organism.The attraction of phlebotomines to hens has been fully established.Lutzomyia longipalpis promptly fed on hens and the abundance of these organisms in henhouses have epidemiologic importance considering VL occurrence 26,27 .
Several authors have previously indicated that the presence of trees, vegetable gardens, plant pots, heaps of wood, leaf and debris build-ups, domestic animals, fowl-breeding activities, animal feces, high contents of organic matter and garbage in soils, and inappropriate wood storage are factors associated to the risk of acquiring leishmaniasis 28,29 .

C
In this study, 204 dogs were analyzed, including animals kept by owners as well as stray dogs captured by the Centro de Controle de Zoonoses, City of Palmas, State of Tocantins, Brazil, by request of R ib e ir ã o Á g u a F ri a UTM -SAD -69 -Zone 22

FIGURE 1 -
FIGURE 1 -Zoning of the City of Palmas, State of Tocantins, Brazil.