First molecular description of autochthonous urban cases of canine visceral leishmaniasis in the city of Belém, Pará, Brazil

Abstract Leishmaniasis is an anthropozoonosis transmitted by vectors, with dogs being the main domestic reservoirs. Brazil is one of the countries most affected by this disease, and it has been described in humans and dogs in every region in the country. In the northern region leishmaniasis cases in humans have been described in more than 100 municipalities in the State, including the capital, Belém. This study involves two cases of canine visceral leishmaniasis in which the animals developed clinical signs compatible with the disease in urban areas in Belém, the Pará state capital. The diagnosis was confirmed via polymerase chain reaction (PCR) to detect SSUr-rDNA and kDNA of Leishmania sp. and Leishmania infantum, respectively. In one of the cases the animal died and in the other the animal underwent treatment with medicines prescribed for dogs. Through this treatment, parasitemia in the second animal has been kept under control and is being monitored through molecular tests. Previously, no canine cases had been notified from urban neighborhoods in the city of Belém, but only on the island of Cotijuba, at a distance of 29 kilometers from the city. Cases of canine and human leishmaniasis have been recorded close to the capital, Belém, which has areas of conserved vegetation and where the presence of disease vectors has been described. Thus, as has been done in several other Brazilian cities, this study uses clinical and laboratory findings to confirm the presence of autochthonous cases of canine visceral leishmaniasis in the city of Belém.


Introduction
Leishmaniasis is a chronic parasitic anthropozoonosis caused by several Leishmania species.Despite being a worldwide public health problem, it is considered a neglected disease related to poverty.Leishmania infects several types of mammals, including marsupials and canines, felines and humans (Gupta et al., 2022).
humans can remain without clinical signs for an extended period (Esteva et al., 2017).
This study describes two cases of autochthonous canine visceral leishmaniasis in the city of Belém (Pará, Brazil) for the first in animals that developed clinical symptoms of the disease and were diagnosed via polymerase chain reaction (PCR).

Study area, Dogs and sample collection
The study was approved by the Animal Use Ethics Committee (AUEC) of the Universidade Federal do Pará (UFPA) with protocol number 20220601.
The first canine case involved a male dog of no defined breed, living in the Umarizal neighborhood in Belém (Figure 1).On the block where this dog lived most houses had wooded backyards, essentially forming a small forest fragment (1°26'13.20"S48°28'51.18"W).The second canine case occurred in June 2020, with a dog of the Pug breed, castrated, age one year and nine months, acquired as a puppy and born and raised in Belém.The guardians lived in the Nazaré neighborhood at a distance of only 600 meters (1°27′18.36″S48°28′.47.18″W) from the Museu Emilio Goeldi Zoobotanical Park (1°27′17.04″S48°28′37.70″W),which includes a preserved forest area with several animals living inside the park, including mammals (Figure 1).
The different Leishmania species cause two main types of clinical forms, cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) (Burza et al., 2018).
Leishmaniasis was initially described as a rural disease.However, over the years, this vector has been adapting to peripheral and urban areas (Rangel and Vilela,, 2008).Such areas often encompass areas with preserved forests and end up becoming transition zones between urban and preserved areas, which facilitates adaptation by the vector.However, the expansion of leishmaniasis is also linked to other factors such as the presence of the main domestic reservoir (dog) (Roque and Jansen, 2014).
In Pará state, northern region of Brazil, leishmaniasis cases in humans have been described in more than 100 municipalities, including the capital, Belém, which, according to SINAN (DATASUS, 2023a, b), reported 231 cases of VL and 3,141 cases of CL in 2020.
In the Americas, dogs are mainly infected by Leishmania infantum, although other Leishmania species from these animals have also been isolated (Alvar et al., 2012, Santos et al., 2020).Dogs develop the disease at a slow rate.The initial clinical signs are skin lesions, alopecia, lymphadenopathy, ulcerative lesions, apathy, anemia, weight loss, and onychogryphosis (Dantas-Torres, 2009).However, skin lesions are generally the most frequent reason for veterinary consultation and the most common finding during physical examination (Saridomichelakis and Koutinas, 2014).Nonetheless, infected dogs as well as Blood samples (5mL) were collected by venipuncture using dry and EDTA-containing tubes for PCR analysis.The nasal and lesion secretions were obtained using sterile swabs on the mucosa.In the first canine case, only the blood sample was sent for molecular evaluation, while in the second case, the veterinarian sent blood, lesion, nasal and secretion samples.The lesion and secretion samples were collected using a sterile cotton swab (Neolab®) and frozen until DNA was extracted.

Extraction of DNA and Molecular detection
For the molecular diagnosis, DNA from the blood samples was extracted using the AxyPrep ™ Blood Genomic DNA Miniprep Kit (Axygen) commercial kit, according to the manufacturer's recommendations.The extraction technique with NaCl (Abrão et al., 2005) was used to extract the DNA from lesions and nasal secretion (swab) with a final volume of 20 μL.After extraction, molecular tests were performed out through PCR targeting the small subunit ribosomal rRNA (SSU rRNA) gene (Uliana et al., 1994;Savani et al., 2005) and extra-chromosomal DNA kinetoplastid DNA (kDNA) (Francino et al., 2006) specific to Leishmania sp and Leishmania infantum, respectively.
For detection to target the small subunit ribosomal rRNA (SSU rRNA) gene (Leishmania sp.) the first PCR was performed with primers S4 (5'-GATCCAGCT GCAGGTTCACC-3') and S12 (5'-GGTTGATTCCGTCAACGGAC-3').Reactions were performed with a final volume of 25 μl containing 1X PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of each primer and 1 U Taq DNA polymerase.DNA was first denatured at 94 o C for 3 min and then cycled 35 times at 94 o C for 1 min, at 50 o C for 1 min, and at 72 o C for 1 min.A final extension of 7 min was performed at 72 o C as described by Uliana et al. (1994).The fragment (520 pb) produced by S4/S12 PCR was used in a nested PCR with primers S17 (5'-CCAAGCTGCC CAGTA GAAT-3') and S18 (5'-TCGGG CGGAT AAAACCC-3'), specific for the genus Leishmania (Savani et al., 2005).The reaction was undertaken under the same conditions as those described above.The S4/S12 PCR product was denatured at 94 o C for 4 min and cycled 30 times; each cycle took place at 94 o C for 1 min, at 55 o C for 1 min, and at 72 o C for 30 s. Oligonucleotides S17 and S18 produced a 490 bp fragment.
Products of small subunit ribosomal rRNA gene (490pb) and kDNA (120 bp) were applied to 1.5% and 2.5% agarose gel, respectively, in Tris-Acetate-EDTA buffer, stained with Diamond Nucleic Acid Dye (Promega Corporation, USA), and later submitted to electrophoresis and visualized under ultraviolet light.An Applied Biosystems thermocycler (Model 2720) was employed for the PCR reactions.

Results
The molecular tests indicated that in the first and second case the animals were infected with Leishmania sp.species L. infantum with the SSUr-rDNA and kDNA markers.In the first case detection in the blood sample, while in the second case it was in eye, nose and lesion secretions.In the first case, although it had never been away from Belém, the animal took walks with its guardian in public squares in Belém, slept outside the house, and did not wear a repellent collar.The main clinical signs observed in the first dog case were apathy, lack of appetite, pale mucous membranes, and exfoliative dermatitis present close to the joints.Subsequently, an ulcerative lesion appeared and a medical evaluation indicated that this might be a case of a lesion caused by Leishmania sp.
In the second canine case the animal developed a lesion in the area close to the snout.A cytological exam of the lesion indicated only an inflammatory process.On physical examination, the animal achieved a good body score, slightly pale oral and ocular mucous membranes, normal lymph nodes, normal rectal temperature (38° C), hypotrichosis in the anterior and posterior limbs, otitis with yellowish discharge in the ears (Malassezia), pododermatitis, papules and crusts in the dorsal region, circular lesion with well-defined edges located on the upper lip that was not itchy and not painful to touch.With the blood count, cell counts were normal, and there was only a slight increase in urea and alkaline phosphatase.
In this case both markers were amplified from the DNA sample extracted from the lesion and ocular and nasal secretion, but not from the blood.The animal began treatment for the disease and began using a repellent spray.After the first month of treatment, the animal showed improvement in the dermatological picture, total regression of the lesion on the face and hair growth in areas with hypotrichosis.A new PCR was performed to detect Leishmania, which was negative in all samples tested (blood and ocular and nasal secretion).Since the negative result, treatment has been maintained continuously to control the disease.

Discussion
This study provides the first autochthonous cases, with clinical presentation and molecular confirmation of two cases of L. infantum infection in dogs in the city of Belém, Pará state, Brazil.
The capital of Belém is known as the city of mango trees.It has extensive tree-lined streets in the center of the city and two zoo-botanical parks, in addition to presenting peripheral areas with conserved vegetation and with springs that supply the city, such as Utinga Park.Studies by Ferreira et al. (2014) described the distribution of leishmaniasis vectors in several preserved areas in the municipality of Belém, including Utinga Park.The enzootic cases of visceral leishmaniasis were restricted to forests surrounding the springs and to the island of Cotijuba (Lainson, 2010).Cotijuba Island, 29 km from Belém, is part of a group of 42 islands that make up the archipelago of Belém and is separated geographically from the capital by the Guajará bay (Figure 1).Human (HL) and canine (CL) leishmaniasis cases have already been described on the island, and epidemiological surveys carried out in the canine population showed a prevalence of 60% of LC, indicating an alarming epidemiological situation for the state and the municipality of Belém (Silveira et al., 2016).
In the case of the state of Pará, leishmaniasis is established in an endemic and epidemic fashion in several municipalities in the state.The existing literature contains studies on cases and the incidence of leishmaniasis in Pará municipalities, including the city of Colares, in northeast Pará (Figueiredo et al., 2014;Galvão, 2018), Marabá and Parauapebas in the southeastern area of the state and Tomé-Açu (Galvão, 2018;Sá, 2019;Santos et al., 2020) and Santarém, in western Pará (Senra et al., 1985).In the Belém metropolitan region, cases have been described on the island of Cotijuba (Silveira et al., 2016) and Barcarena (Silveira et al., 2012;Lima et al., 2010).Galvão (2018) recorded a frequency of 37.7% (23/49) in biological samples from dogs residing in the city of Belém, but in none of them was it possible to prove an autochthonous case.This was because all the infected dogs had traveled to other endemic cities in Pará at some point or were animals that had previously resided in other Brazilian municipalities where human and canine cases have been described.
Several studies in other Brazilian cities have described autochthonous cases of CL (Tolezano et al., 2007;Silva et al., 2011;Faria et al., 2017, Silva et al., 2018;Rocha et al., 2018, Pinto et al., 2021).Furthermore, although animals from Belém have been diagnosed with leishmaniasis (antibody detection) in two other studies (Campos et al., 2017, Schwanke et al., 2014) neither study indicated that the animals were born in Belém and never lived or traveled to endemic areas, as described for the animals in this study.The study by Schwanke et al. (2014) failed to amplify the L. infantum DNA in the samples of dogs tested, and its results are based on serological data.Thus, based on the clinical symptoms and life histories of the animals, as well as the molecular diagnosis procedures described in this study, these are the first confirmed autochthonous reports of CL caused by L. infantum in two neighborhoods in the city Belém, Pará (Figure 1).
Detection of canine infection is an important public health indicator since it most often precedes human infection (Silva et al., 2018).A differential diagnosis of CL is indispensable, especially in areas where there are human cases described, given that the domestic dog is considered the main reservoir of the disease in urban areas.Molecular diagnosis is advantageous over other methods because it has high specificity and sensitivity and can be applied to different types of biological samples.The widely used serological tests can fail in dogs with prepatent infections that may return to seronegativity, even in those where the immune response was not able to make the seroconversion to IgG (Coura-Vital et al., 2013).
Given that mammals (Cerdocyon thous) infected with Leishmania infantum have been found inside preserved areas in Belém (Utinga Park) and infected dogs have been found in localities close to the Capital (Ilha de Cotijuba e Barcarena).There is a need for setting up surveillance and control inside the city, as recommended by the Brazilian Ministry of Health of Brazil (Brasil, 2011(Brasil, , 2014)), to track developments in neighborhoods with possible infections, as was done in this study, given that Belém has ideal temperature and humidity conditions for sandflies to develop.(Silveira et al., 2012).Besides our study, other researchers (Trüeb et al., 2018;Silva et al., 2022) have shown that urbanized areas present differences in sandfly richness and diversity compared to forest fragments, and have concluded that the conservation of forest areas, even as fragments in urban areas, favors the diversity of phlebotomine species.As has been done in several other Brazilian cities, this study uses clinical and laboratory findings to confirm the presence of autochthonous cases of canine visceral leishmaniasis in an urban area in the city of Belém.

Figure 1 .
Figure 1.Location of two cases of autochthonous canine leishmaniasis in the city of Belém, Para (Brazil).