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Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil
Veterinary Parasitology 176 (2011) 281–282
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil Willian Marinho Dourado Coelho a , Virgínia Bodelão Richini-Pereira b , Helio Langoni b , Katia Denise Saraiva Bresciani a,∗ a
Departamento de Apoio, Produc¸ão e Saúde Animal, FOA, UNESP, Rua Clóvis Pestana, 793 CEP: 16050-680, Arac¸atuba, São Paulo, Brazil Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia – UNESP, Distrito Rubião Jr., s/n CEP: 18618-970, Botucatu, São Paulo, Brazil
b
a r t i c l e
i n f o
Article history: Received 10 June 2010 Received in revised form 27 October 2010 Accepted 28 October 2010 Keywords: Cat Leishmania (L.) chagasi PCR
a b s t r a c t The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, São Paulo State. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Infection by Leishmania spp. in cats has been reported in several countries (Petersen, 2009; Tabar et al., 2008), including Brazil (da Silva et al., 2008; Savani et al., 2004; Serrano et al., 2008). The aim of this work was to molecularly detect Leishmania spp. specimens obtained from cats (Felix catus) in Andradina Municipality, São Paulo State, Brazil. The experimental group constituted of 52 cats of both sexes, several breeds and age ranges, sent by their owners to the Center for Zoonosis Control at Andradina Municipality, São Paulo State, Brazil, for euthanasia. This municipality is located in Arac¸atuba Mesoregion, São Paulo State (20.8961◦ , 51.37944◦ ), at 405 m altitude. This procedure was previously approved by the Ethics Committee on
∗ Corresponding author. Tel.: +55 18 37227505. E-mail addresses: [email protected] (W.M.D. Coelho), [email protected] (K.D.S. Bresciani). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.052
Animal Experimentation of Arac¸atuba School of Dentistry (FOA), UNESP, protocol no. 2007-003276. Fragments of popliteal lymph nodes, spleen and femoral bone marrow were collected for imprint and were stained with Rapid Panoptic (Hematocor, Biolog® ) to allow the visualization of amastigote forms of Leishmania spp. by observing 300 fields at 1000× magnification. DNA was extracted by means of a commercial kit (Genomic PrepTM Cells and Tissue DNA GE Healthcare). PCR reaction, of kinetoplast minicircle DNA, was performed in a final volume of 25 l using the following reagents: 10 mM Tris–HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM dNTPs, 10 pmol of each primer RV1 and RV2 (Lachaud et al., 2002), 0.2 units of Platinum Taq DNA polymerase (Invitrogen, Brazil) and 10 ng DNA template. The amplification mixture was incubated in a Mastercycler Gradient (Eppendorf) by using the following cycling profile: initial denaturation at 94 ◦ C for 4 min, 40 cycles of 94 ◦ C for 30 s, 59 ◦ C for 30 s, 72 ◦ C for 30 s, and 70 ◦ C for 10 min. The amplified products were analyzed by electrophoresis on 1.5% agarose gel stained with SYBR® safe (Invitrogen® ). The amplified DNA frag-
282
W.M.D. Coelho et al. / Veterinary Parasitology 176 (2011) 281–282
lar identities of the amplicons were confirmed by direct double-strand sequencing which showed 97% similarity with Leishmania (L.) chagasi DNA sequences deposited at GenBank (access number Z35276). The positivity obtained in our study (5.76%) was similar to the results of Rossi (2007) in Arac¸atuba, evidencing 6.5% (13/200) cats infected with Leishmania spp. Considering other countries, the percentage obtained in epidemiological surveys was 30.4% (7/23) in Portugal by PCR (Maia et al., 2008). In our study, there were 3.84% (2/52) positive samples in lymph node imprint. Rossi (2007), in Arac¸atuba, verified 4% (8/200) positivity for Leishmania spp. in aspiration biopsies of lymph nodes, bone marrow, spleen and liver. The results obtained in this study led to the characterization of infection by Leishmania (L.) chagasi in felines from Andradina Municipality, São Paulo State, where there is high incidence of leishmaniasis in dogs and eventually in humans. References
Fig. 1. Electrophoresis of the PCR products using primers RV1 and RV2 with products of 150 pb in samples of spleen and right and left popliteal lymph node of two cats from Andradina municipality, São Paulo state, Brazil.
ments were visualized in an image analyzer (GelDoc-ITTM Imaging System), using Vision Works® LS Software. The amplicons were purified with ExoSap-IT (USB) and sequencing reactions were performed for both strands according to the protocol of DYEnanic ET Dye termination kit (GE Healthcare) and run on a MegaBaceTM 1000 (GE-Healthcare). The sequences were compared to those in NCBI database by using BLASTn (Basic Local Alignment Tool for Nucleotide). 2. Discussion Infection by Leishmania spp. was detected for 5.76% (3/52) animals analyzed by means of parasitological and molecular techniques. The amplification of Leishmania (L.) chagasi was positive for spleen and lymph node samples from two symptomatic animals (Fig. 1). The molecu-
da Silva, A.V., de Souza Cândido, C.D., de Pita Pereira, D., Brazil, R.P., Carreira, J.C., 2008. The first record of American visceral leishmaniasis in domestic cats from Rio de Janeiro Brazil. Acta Trop. 105, 92– 94. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Maia, C., Nunes, M., Campino, l., 2008. Importance of cats in zoonotic leishmaniasis in Portugal. Vector Borne Zoonotic Dis. 8, 555– 559. Petersen, C.A., 2009. Leishmaniasis, an emerging disease found in companion animals in the United States. Top. Comp. Anim. Med. 24, 182– 188. Rossi, C.N., 2007. Ocorrência de Leishmania sp. em gatos do município de Arac¸atuba – São Paulo – Brasil. Dissertac¸ão apresentada à Faculdade de Ciências Agrárias e Veterinárias–UNESP, campus de Jaboticabal, como parte das exigências para obtenc¸ão do título de Mestre em Medicina Veterinária (Clínica Médica Veterinária). Savani, E.S.M.M., Camargo, M.C.G.O., Carvalho, M.R., Zampieri, R.A., Santos, M.G., D’auria, S.R.N., Shaw, J.J., Floeter-Winter, L.M., 2004. The first record in the Americas of an autochthonous case of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felix catus) from Cotia County, São Paulo State Brazil. Vet. Parasitol. 120, 229– 233. Serrano, A.C.M., Nunes, C., Savani, E.S.M., D’auria, S.R.N., Bonello, F.L., Vasconcelos, R.O., Lima, V.M.F., Bresciani, K.D.S., 2008. Leishmaniose em felino na zona urbana de Arac¸atuba – SP – relato de caso. Clín. Vet. 12, 36–40. Tabar, M.D., Altet, L., Francino, O., Sanchez, A., Ferrer, L., Roura, X., 2008. Vector-borne infection in cats: molecular studies in Barcelona area (Spain). Vet. Parasitol. 151, 332–336.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil Willian Marinho Dourado Coelho a , Virgínia Bodelão Richini-Pereira b , Helio Langoni b , Katia Denise Saraiva Bresciani a,∗ a
Departamento de Apoio, Produc¸ão e Saúde Animal, FOA, UNESP, Rua Clóvis Pestana, 793 CEP: 16050-680, Arac¸atuba, São Paulo, Brazil Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia – UNESP, Distrito Rubião Jr., s/n CEP: 18618-970, Botucatu, São Paulo, Brazil
b
a r t i c l e
i n f o
Article history: Received 10 June 2010 Received in revised form 27 October 2010 Accepted 28 October 2010 Keywords: Cat Leishmania (L.) chagasi PCR
a b s t r a c t The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, São Paulo State. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Infection by Leishmania spp. in cats has been reported in several countries (Petersen, 2009; Tabar et al., 2008), including Brazil (da Silva et al., 2008; Savani et al., 2004; Serrano et al., 2008). The aim of this work was to molecularly detect Leishmania spp. specimens obtained from cats (Felix catus) in Andradina Municipality, São Paulo State, Brazil. The experimental group constituted of 52 cats of both sexes, several breeds and age ranges, sent by their owners to the Center for Zoonosis Control at Andradina Municipality, São Paulo State, Brazil, for euthanasia. This municipality is located in Arac¸atuba Mesoregion, São Paulo State (20.8961◦ , 51.37944◦ ), at 405 m altitude. This procedure was previously approved by the Ethics Committee on
∗ Corresponding author. Tel.: +55 18 37227505. E-mail addresses: [email protected] (W.M.D. Coelho), [email protected] (K.D.S. Bresciani). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.052
Animal Experimentation of Arac¸atuba School of Dentistry (FOA), UNESP, protocol no. 2007-003276. Fragments of popliteal lymph nodes, spleen and femoral bone marrow were collected for imprint and were stained with Rapid Panoptic (Hematocor, Biolog® ) to allow the visualization of amastigote forms of Leishmania spp. by observing 300 fields at 1000× magnification. DNA was extracted by means of a commercial kit (Genomic PrepTM Cells and Tissue DNA GE Healthcare). PCR reaction, of kinetoplast minicircle DNA, was performed in a final volume of 25 l using the following reagents: 10 mM Tris–HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM dNTPs, 10 pmol of each primer RV1 and RV2 (Lachaud et al., 2002), 0.2 units of Platinum Taq DNA polymerase (Invitrogen, Brazil) and 10 ng DNA template. The amplification mixture was incubated in a Mastercycler Gradient (Eppendorf) by using the following cycling profile: initial denaturation at 94 ◦ C for 4 min, 40 cycles of 94 ◦ C for 30 s, 59 ◦ C for 30 s, 72 ◦ C for 30 s, and 70 ◦ C for 10 min. The amplified products were analyzed by electrophoresis on 1.5% agarose gel stained with SYBR® safe (Invitrogen® ). The amplified DNA frag-
282
W.M.D. Coelho et al. / Veterinary Parasitology 176 (2011) 281–282
lar identities of the amplicons were confirmed by direct double-strand sequencing which showed 97% similarity with Leishmania (L.) chagasi DNA sequences deposited at GenBank (access number Z35276). The positivity obtained in our study (5.76%) was similar to the results of Rossi (2007) in Arac¸atuba, evidencing 6.5% (13/200) cats infected with Leishmania spp. Considering other countries, the percentage obtained in epidemiological surveys was 30.4% (7/23) in Portugal by PCR (Maia et al., 2008). In our study, there were 3.84% (2/52) positive samples in lymph node imprint. Rossi (2007), in Arac¸atuba, verified 4% (8/200) positivity for Leishmania spp. in aspiration biopsies of lymph nodes, bone marrow, spleen and liver. The results obtained in this study led to the characterization of infection by Leishmania (L.) chagasi in felines from Andradina Municipality, São Paulo State, where there is high incidence of leishmaniasis in dogs and eventually in humans. References
Fig. 1. Electrophoresis of the PCR products using primers RV1 and RV2 with products of 150 pb in samples of spleen and right and left popliteal lymph node of two cats from Andradina municipality, São Paulo state, Brazil.
ments were visualized in an image analyzer (GelDoc-ITTM Imaging System), using Vision Works® LS Software. The amplicons were purified with ExoSap-IT (USB) and sequencing reactions were performed for both strands according to the protocol of DYEnanic ET Dye termination kit (GE Healthcare) and run on a MegaBaceTM 1000 (GE-Healthcare). The sequences were compared to those in NCBI database by using BLASTn (Basic Local Alignment Tool for Nucleotide). 2. Discussion Infection by Leishmania spp. was detected for 5.76% (3/52) animals analyzed by means of parasitological and molecular techniques. The amplification of Leishmania (L.) chagasi was positive for spleen and lymph node samples from two symptomatic animals (Fig. 1). The molecu-
da Silva, A.V., de Souza Cândido, C.D., de Pita Pereira, D., Brazil, R.P., Carreira, J.C., 2008. The first record of American visceral leishmaniasis in domestic cats from Rio de Janeiro Brazil. Acta Trop. 105, 92– 94. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Maia, C., Nunes, M., Campino, l., 2008. Importance of cats in zoonotic leishmaniasis in Portugal. Vector Borne Zoonotic Dis. 8, 555– 559. Petersen, C.A., 2009. Leishmaniasis, an emerging disease found in companion animals in the United States. Top. Comp. Anim. Med. 24, 182– 188. Rossi, C.N., 2007. Ocorrência de Leishmania sp. em gatos do município de Arac¸atuba – São Paulo – Brasil. Dissertac¸ão apresentada à Faculdade de Ciências Agrárias e Veterinárias–UNESP, campus de Jaboticabal, como parte das exigências para obtenc¸ão do título de Mestre em Medicina Veterinária (Clínica Médica Veterinária). Savani, E.S.M.M., Camargo, M.C.G.O., Carvalho, M.R., Zampieri, R.A., Santos, M.G., D’auria, S.R.N., Shaw, J.J., Floeter-Winter, L.M., 2004. The first record in the Americas of an autochthonous case of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felix catus) from Cotia County, São Paulo State Brazil. Vet. Parasitol. 120, 229– 233. Serrano, A.C.M., Nunes, C., Savani, E.S.M., D’auria, S.R.N., Bonello, F.L., Vasconcelos, R.O., Lima, V.M.F., Bresciani, K.D.S., 2008. Leishmaniose em felino na zona urbana de Arac¸atuba – SP – relato de caso. Clín. Vet. 12, 36–40. Tabar, M.D., Altet, L., Francino, O., Sanchez, A., Ferrer, L., Roura, X., 2008. Vector-borne infection in cats: molecular studies in Barcelona area (Spain). Vet. Parasitol. 151, 332–336.