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.

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“…The clinical signs of leishmaniasis are directly related to the immune response of the infected dog and we can account the disease into four stages based on serological status, clinical signs, laboratory findings and type of therapy and prognosis for each stage [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Of course, these aspects are result of LEVs interactions with the hosts.…”

“…However, for the greater reliability of the results, PCR is indicated for various types of samples, given the importance of the Leishmania infection, mainly due to its high zoonotic potential. Detection of infection by amplifying kDNA segments and SSUrrDNA showed similar sensitivity, indicating that both can be used with for the diagnosis of canine leishmaniasis [29,30]. The sequencing of 18S rRNA segments allowed the identification of L. infantum in all cases, and four animals were infected by strains with genetic variability, with three heterozygous samples and one with one with one pyrimidine (C→T) transition-type mutation.…”

“…Following Galvão et al tested protocols, 5mL of canine blood samples were collected by venipuncture using dry and EDTA-containing tubes for PCR analysis and the secretions from auditive duct, mucous membrane of the anus, oral mucosa, whole blood, preputial and ocular of "Jimmy" were obtained using sterile cotton swab (Neolab ® ) and frozen until DNA was extracted (Preliminary studies = blood samples and ocular swabs from 130 dogs: 67 males (51.5%), 60 females (46.1%) and three animals (2.4%) had no gender information, adding the samples from "Jimmy" the final total = 131 animals from the endemic areas of State of Pará-Amazon Region-Brazil -area: 1.284.042 km, situated between the coordinates 04°20′45″ N and 09°50′27″ S, and 46°03′18″) [29,30]. The extraction of DNA for the molecular diagnosis from the canine blood samples was made according to the manufacturer's recommendations of AxyPrep ™ Blood Genomic DNA Miniprep Kit (Axygen) commercial kit and the technique with NaCl to extract the DNA from secretions by swabs (final volume of 20 μL).…”

“…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. And for the PCR reactions an Applied Biosystems thermocycler (Model 2720) was employed [29,30]. After obtaining the amplicons, the PCR products were purified for the kDNA gene and nested-PCR for the SSU-rDNA gene using EXO-SAP PCR DNA (GE Healthcare), in the Sequencing was performed with the aid of the ABI 3500XL automatic DNA analyzer Applied Biosystems ™ in combination with BigDye ® Terminator v3.1.…”

“…Relationships phylogenetic studies were constructed with Neighbor-Joining, the p distance method was used to calculate the genetic distances, the monophyletic groups have been supported by the Bootstrap 2000 method, using MEGA 6 program. And to test whether a relationship exists between the diagnostic methods exists by a contingency table the BioEstat 5.0 software was used for results considered as relevants (p<0.05) [29,30].…”