Evaluation of Leishmania ( Leishmania ) chagasi strains isolated from dogs originating from two visceral leishmaniasis-endemic areas in Brazil using multilocus enzyme electrophoresis

Introduction: Domestic dogs are the most important reservoir in the peridomestic transmission cycle of Leishmania (Leishmania) chagasi. The genetic variability of subpopulations of this parasite circulating in dogs has not been thoroughly analyzed in Brazil, even though this knowledge has important implications in the clinical-epidemiological context. Methods: The objective of this study was to evaluate and compare the phenotypic variability of 153 L. chagasi strains isolated from dogs originating from the municipalities of Rio de Janeiro (n = 57) and Belo Horizonte (n = 96), where the disease is endemic. Strains isolated only from intact skin were selected and analyzed by multilocus enzyme electrophoresis using nine enzyme systems (6PG, GPI, NH1 and NH2, G6P, PGM, MDH, ME, and IDHNADP). Results: The electrophoretic profile was identical for all isolates analyzed and was the same as that of the L. chagasi reference strain (MHOM/BR/74/PP75). Phenetic analysis showed a similarity index of one for all strains, with the isolates sharing 100% of the characteristics analyzed. Conclusions: The results demonstrate that the L. chagasi populations circulating in dogs from Rio de Janeiro and Belo Horizonte belong to a single zymodeme.


Palavras
In Brazil, Leishmania (Leishmania) chagasi [syn.Leishmania infantum] is the only agent that causes visceral leishmaniasis and is associated with the vector Lutzomyia longipalpis in most regions.Recently, Leishmania (Leishmania) chagasi has been detected in natural canine infections in areas unaffected by visceral leishmaniasis 4 .The role of the dog as a domestic reservoir in the transmission cycle of visceral leishmaniasis has been well established.The disease progresses slowly in this animal but shows an insidious onset and a variable spectrum of clinical manifestations.During more advanced stages of the disease, onychogryphosis, splenomegaly, lymphadenopathy, alopecia, dermatitis, skin ulcers, keratoconjunctivitis, a runny nose, anemia, apathy, diarrhea, intestinal hemorrhage, paw edema, vomiting, and hyperkeratosis are frequent findings 5 .However, the disease may remain latent in some cases and may even resolve spontaneously 6 .
The identity of the etiological agent of visceral leishmaniasis in the Americas has been the matter of numerous discussions [7][8][9] .For a long time, L. chagasi has been considered to be an autochthonous agent in the New World, and this hypothesis directed studies that genetically compared L. chagasi and L. infantum using isolates obtained from different hosts and by different methods [10][11][12][13] .In all studies cited, L. chagasi and L. infantum presented similar genetic profiles and were considered to be the same species.It is currently assumed that this parasite was introduced to South America during European colonization through dogs infected with L. infantum, the predominant species in Mediterranean countries, and that L. infantum adapted to new vectors and hosts 14,15 .

Countinho CER et al -Evaluation of L. chagasi strains by MLEE
The correct classification of Leishmania parasites is fundamental for the establishment of control measures for the diseases caused by these organisms, and numerous tools are currently applied for this purpose.In this respect, multilocus enzyme electrophoresis (MLEE) is the reference standard for the identification of species of the genus Leishmania and is one of the methods most used for the characterization of species of this genus [16][17][18][19][20][21] .The characterization of isolates is important, especially in epidemiological surveillance studies, permitting the mapping of Leishmania species and variants that circulate in humans and animals in a given region.In addition, this characterization contributes to a better understanding of the factors related to these phenotypic variations, such as clinical characteristics, drug resistance, parasite virulence, preference for certain hosts, and association with certain immunosuppressive diseases.
In view of the importance of the domestic dog in the transmission cycle of L. chagasi and the importance of studies investigating the genetic variability of isolates circulating in this host, the objective of the present investigation was to evaluate and compare the phenotypic characteristics of 153 Leishmania strains isolated from dogs originating from two endemic areas in Brazil.
The 153 isolates studied were obtained from the strain collection of the Laboratory of Leishmaniasis Surveillance (VigiLeish, IPEC/ FIOCRUZ).The strains isolated from dogs were obtained during epidemiological surveys carried out in the municipalities of Rio de Janeiro (Campo Grande, Guaratiba, Barra de Guaratiba, Barra da Tijuca, Jacarepaguá, Realengo and Bangu) during the period from 2000 to 2008 (RJ; n= 57) and Belo Horizonte (Barreiro, Centro Sul, Leste, Nordeste, Noroeste, Norte, Oeste, Pampulha and Lagoa de Pampulha) in 2004 (BH; n = 96).Both regions are located in southeastern Brazil.Only strains isolated from the intact skin of dogs presenting different clinical conditions were selected.First, the isolates were recovered from liquid nitrogen storage and expanded in Schneider's medium (Sigma) supplemented with 10% fetal bovine serum (Cultilab) and antibiotics (200 units/mL penicillin G potassium and 100 units/mL streptomycin) to obtain approximately 10 9 parasites.When in the exponential phase, the culture was pelleted by centrifugation, resuspended in appropriate buffer, and stored at -196ºC until isoenzyme electrophoresis.
The MLEE bands were compared using the simple matching coefficient of similarity, and the matrix was transformed into a dendrogram using the UPGMA algorithm 23 .Phenetic analysis was performed with the NTSYS-pc program, version 2.02 (Exeter Software, Setauket, NY, USA).

Ethical considerations
The collection of biological samples from the dogs was approved by the Ethics Committee on the Use of Animals of FIOCRUZ (CEUA/FIOCRUZ), license numbers P-286/06 and L-023/06.All 153 canine isolates analyzed by MLEE demonstrated similar electrophoretic profiles, which were similar to that of the L. chagasi reference strain (MHOM/BR/74/PP75).No isoenzyme variants were observed among parasites isolated from Rio de Janeiro and Belo Horizonte.All isolates were classified into the same zymodeme, with a similarity index of one, demonstrating that all isolates studied shared 100% of the characteristics analyzed (Figure 1).Visceral leishmaniasis is widely distributed in Brazil, with outbreaks occurring in urban areas, a fact highlighting the importance of the domestic dog in this context 2,24,25 .Clinically, dogs infected with L. chagasi can show a broad spectrum of manifestations or can remain asymptomatic, suggesting the circulation of distinct subpopulations of the parasite in these hosts.Because of this variation, studies investigating the genetic variability of strains isolated from dogs are extremely important but are scarce in the literature.Therefore, the objective of this study was to evaluate and compare the phenotypic characteristics of L. chagasi strains isolated from dogs originating from two endemic areas in Brazil.
In addition to being an effective method for the identification of Leishmania isolates, isoenzyme electrophoresis has various applications.One of the disadvantages of this technique is the need for previous isolation of the parasite to subsequently obtain a large culture volume, a fact that has contributed to the use of PCR-based methods in many studies 26,27 .However, the variability of parasites of the genus Leishmania has been widely studied by MLEE 11,19,28 .
In the present study, nine enzyme systems were used for the analysis of 153 L. chagasi strains isolated from animals originating from two endemic regions.These isolates presented identical electrophoretic profiles.This result demonstrates that the isolates originating from the two endemic regions showed no difference that could be associated with geographic area or host status, indicating -chaves: Cão.Leishmania chagasi.Variabilidade genética.MLEE.

FIGURE 1 -
FIGURE 1 -MLEE dendrogram showing the similarity among the canine samples obtained from two areas endemic for visceral leishmaniasis and the Leishmania chagasi reference strain.