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Leishmania chagasi in dogs from the city of Jaciara, Mato Grosso, Brazil/Leishmania chagasi em caes no municipio de Jaciara, Mato Grosso, Brasil.

Visceral leishmaniasis (VL) is a zoonotic disease that is transmitted by sand flies and caused by L. chagasi (syn. L. infantum) in the Americas. The dog is considered the primary reservoir of the aetiologic agent of VL, and dogs have an important role in maintaining the disease transmission cycle in urban areas because of the parasitism of their skin, regardless of the dogs clinical presentation and proximity to man (BRASIL, 2006; BRITO et al., 2014a, b).

Various tissues, such as blood, skin, spleen, bone marrow, liver, and lymph nodes, have been identified as good sites for the parasitological confirmation of canine visceral leishmaniasis (CVL) (LACHAUD et al., 2002; ALMEIDA et al., 2013; RAMOS et al., 2013). The ease of obtaining the sample, the clinical status of the animal and the laboratory technique should be considered for the diagnosis (MARTINEZ et al., 2011). In accordance to this list of considerations, the Polymerase Chain Reaction has emerged as an important alternative diagnostic technique (LACHAUD et al., 2002; GOMES et al., 2007), which has adequate sensitivity to detect the DNA of the agent in host tissue prior to the occurrence of seroconversion, providing an early diagnosis of the infection (REIS et al., 2013).

In areas of sympatry with other trypanosomatid agents, the identification of circulating Leishmania species in dogs is an important component in the epidemiological study of leishmaniasis, and diagnosis by PCR has been used for this purpose (ALMEIDA et al., 2013). The municipality of Jaciara has presented VL cases since 2003 with a prevalence of 54.7% CVL (BRITO et al., 2014a; 2014b; LOPES et al., 2014) without the confirmation of Leishmania species in dogs. In this context, the aim of this study was to identify the Leishmania species in domestic dogs in the urban Jaciara area by PCR and to evaluate the use of this diagnostic technique in different tissues.

This study was conducted in the municipality of Jaciara (15 57' 22" S and 54 57' 48" W), which is located in the Cerrado of Mato Grosso (IBGE, 2014). The dogs (which were of different breeds, ages and sexes and lived in the urban area) that were identified as seropositive by Enzyme-Linked Immunosorbent Assay (ELISA) and Indirect Immunofluorescence Reaction (IFA) as part of a routine public health screening were forwarded to the Municipal Health Department for euthanasia.

The evaluated dogs were clinically classified as symptomatic, asymptomatic or oligosymptomatic (ALMEIDA et al., 2013). Blood samples were collected by venipuncture (cephalic or jugular). After the euthanasia (CFMV, 2012), bone marrow (the region of the iliac crest), skin fragments (scapular region) and spleen fragments were collected.

The sample DNA was extracted according to GOMES et al. (2007) by phenol/chloroform method. The PCR primers 150 and 152 were used for Leishmania sp. (DEGRAVE et al., 1994) and RV1 and RV2 (LACHAUD et al., 2002) were used for L. chagasi. A DNA reference strain of L. infantum (MHOM / BR / 1974 / PP75) was used as positive control, and a DNA-free reaction was employed as negative control. Amplified products were fractionated by electrophoresis on a 2% agarose gel, stained with red gel and visualized by UV transillumination.

The data were analysed with Epi Info software 3.3.2 (CDC, Atlanta, USA) by chi-square or Fisher's exact tests to assess the association between the presence of Leishmania sp. DNA in different sampled tissues and the clinical status of the dogs, with a significance level of 5%.

Of the 101 dogs evaluated by PCR for Leishmania sp., parasite DNA was detected in the samples from 97 dogs (96.03%) and identified as L. chagasi. In analysing the collected tissues, DNA amplification occurred in 92 (91.08%) spleen samples, 78 (78%) bone marrow samples, 69 (68.31%) skin samples and 45 (44.55%) blood samples (Table 1). In comparison with positive results between tissues, the spleen was statistically superior to Leishmania DNA detection in bone marrow (P=0.003, OR=9.38 [1.80 to 54.04]. The marrow was statistically superior to the skin for detection (P=0.004; OR=4.49 [1.50 to 13.70]). There was no significant difference among the other tissues (p>0.05).

In considering the clinical condition of the dogs, 1 (0.99%) was rated asymptomatic, 14 (13.86%) were oligosymptomatic and 86 (85.14%) were symptomatic. Of these dogs, 01 (100%), 12 (85.71%) and 84 (97.67%) were positive according to the PCR.

Several regions in Brazil are endemic for CVL, with Jaciara being one of the cities with the highest serological prevalence in Mato Grosso (BRITO et al., 2014a; LOPES et al., 2014). The observation that 96.04% of PCR positives were associated with seropositive dogs confirms the occurrence of the infection, despite of the possibility of cross-reactivity in serological tests as recommended by the Ministry of Health (BRAZIL, 2006; MADEIRA et al., 2009). This finding also enabled the identification of the L. chagasi species in domestic reservoirs in Jaciara, which were previously found only in the vector (BRITO et al., 2014b).

Although there is variability in the PCR results because of the type of primers used and the target of the amplified DNA (ALMEIDA et al., 2013; REIS et al., 2013), in the present study it was used the kinetoplast DNA sequences to increase the test sensitivity by presenting a large number of parasite copies (REIS et al., 2013), which combined the use of different samples to allow a higher positivity rate.

Previous studies described an increased percentage of L. chagasi DNA detection in symptomatic dogs (ALMEIDA et al., 2013), a finding that was not observed in this study. However, this discrepancy should be analysed with caution because the symptomatic dogs were numerically superior to and olygosymptomatic and asymptomatic, preventing a reliable statistical analysis. In this study, skin, bone marrow, blood and spleen in agreement with previous studies (MADEIRA et al., 2009; ALMEIDA et al., 2013), and the spleen showed a higher percentage of L. chagasi DNA detection than the bone marrow. According to REIS et al. (2013), the spleen is considered one of the major organs affected by Leishmania sp.; however, its invasive collection hampers its applicable at a large scale. The superiority of detecting the infection in bone marrow compared with skin differs from that described by ALMEIDA et al. (2013). However, the positive difference between the sampled tissues may be related to Leishmania's tropism for lymphoid organs (RAMOS et al., 2013).

In summary, the spleen presented the strongest detection of Leishmania confirmed as Leishmania chagasi, followed by bone marrow tissue, emphasizing the importance of aetiological identification in areas with sympatric species.

http://dx.doi.org/10.1590/0103-8478cr20150126

ACKNOWLEDGEMENTS

This study was supported by the following grant: Universal 2011-Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) (AUXPE no 3531/2014).

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

The consent of the dog owners was obtained and indicated by signatures on the science terms/euthanasia agreement, and the procedures were approved by the Ethics Committee on Animal Use at the Universidade Federal de Mato Grosso (CEUA/ UFMT) under protocol number 23108.018081/12-0.

REFERENCES

ALMEIDA, A.B.P.F. et al. Canine visceral leishmaniasis: diagnostic approaches based on polymerase chain reaction employing different biological samples. Diagnostic Microbiology and Infectious Disease, v.76, p.321-324, 2013. Available from: <http://dx.doi.org/10.1016/j.diagmicrobio.2013.03.017>. Accessed: Dec. 20, 2013. doi: 10.1016/j.diagmicrobio.2013.03.017.

BRASIL. Ministerio da Saude. Departamento de Vigilancia Epidemiologica. Secretaria de Vigilancia em Saude. Manual de vigilancia e controle da leishmaniose visceral. Secretaria de Vigilancia em Saude. Brasilia: Ministerio da Saude, 2006. 120p. Available from: <http://portal.saude.gov.br/portal/arquivos/pdf/manual_leish_visceral2006.pdf>. Accessed: Dec. 20, 2013.

BRITO, V.N. et al. Epidemiological aspects leishmaniasis in Jaciara, Mato Grosso, Brazil, 2003 to 2012. Revista Brasileira de Parasitologia Veterinaria, v.23, n.1, p.63-68, 2014a. Available from: <http://dx.doi.org/10.1590/S1984-29612014008>. Accessed: Sept. 17, 2014. doi: 10.1590/S1984-29612014008.

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LOPES, P.M. et al. Seroprevalence and risk factors associated with visceral leishmaniasis in dogs in Jaciara, State of Mato Grosso. Revista da Sociedade Brasileira de Medicina Tropical, v.47, n.6, p.791-795, 2014. Available from: <http://dx.doi.org/10.1590/0037-8682-0027214>. Accessed: Jan. 20, 2015. doi: 10.1590/0037-8682-0027-214.

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Patricia Lazari (I) Arleana do Bom Parto Ferreira de Almeida (II) Valeria Dutra (II) Luciano Nakazato (II) Alvaro Felipe de Lima Ruy Dias (I) Veruska Nogueira de Brito (III) Cenita Maria Oliveira (IV) Valeria Regia Franco Sousa (II) *

(I) Programa de Pos-graduacao em Ciencias Veterinarias, Faculdade de Agronomia, Medicina Veterinaria e Zootecnia (FAMEVZ), Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brasil.

(II) Departamento de Clinica Medica Veterinaria, FAMEVZ, UFMT, Cuiaba, MT, Brasil. E-mail: regia@ufmt.br. * Corresponding author.

(III) Vigilancia Ambiental em Saude, Secretaria Estadual de Saude de Mato Grosso, MT, Brasil.

(IV) Vigilancia Ambiental em Saude, Secretaria de Saude de Jaciara, MT, Brasil.

Received 01.31.15 Approved 06.25.15 Returned by the author 09.11.15 CR-2015-0126.R2
Table 1--Leishmania chagasi detection by PCR in different
tissues according to clinical status of dogs.

                              Sampled Tissues

                     Spleen      Bone Marrow    Skin
Clinical Status      (n=101)     (n=100)        (n=101)

Asymtomatic (n=01)   01          01             01
Oligosymptomatic     12          11             08
  (n=14)
Symtomatic (n=86)    79          66             60
TOTAL (%)            92 (91,1)   78 (78)        69 (68,3)

                         Sampled Tissues

                     Blood
Clinical Status      (n=101)     +/n (%)

Asymtomatic (n=01)   00          01/01 (100)
Oligosymptomatic     04          12/14 (85,7)
  (n=14)
Symtomatic (n=86)    41          84/86 (97,7)
TOTAL (%)            45 (44,5)   97/101 (96)

+ - total number of positive dogs, n- sampling.
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Title Annotation:parasitologia; texto en ingles
Author:Lazari, Patricia; de Almeida, Arleana do Bom Parto Ferreira; Dutra, Valeria; Nakazato, Luciano; Dias
Publication:Ciencia Rural
Date:Feb 1, 2016
Words:2082
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