Molecular and parasitological detection of Leishmania spp. in a dipteran of the species Tabanus importunus

Detecção molecular e parasitológica de Leishmania spp. em díptero da espécie Tabanus importunus

Willian Marinho Dourado Coelho Katia Denise Saraiva Bresciani About the authors

Abstracts

Leishmaniasis is an important chronic zoonosis caused by protozoa of the genus Leishmania spp. The major vectors of this protozoosis are sand flies, and Lutzomyia longipalpis is considered the main species implicated in the transmission of American Visceral Leishmaniasis in Brazil. The presence of the parasite's deoxyribonucleic acid (DNA) in ectoparasites such as ticks and fleas has prompted speculations about the existence of new vectors in the cycle of leishmaniasis. The aim of this paper is to report the molecular detection of Leishmania spp. in a horse fly of the species Tabanus importunus which parasitized an oligosymptomatic dog infected with Leishmania spp. Molecular amplification of the protozoan's DNA in the head, thoracic region and abdomen of the tabanid tested positive for Leishmania complex. This is the first report of the presence of DNA from Leishmania spp. in dipterous insects of the species T. importunus.

Dogs; canine visceral leishmaniasis; horse fly; PCR; protozoan; vectors


A leishmaniose é uma importante zoonose, de caráter crônico, causada por protozoários do gênero Leishmania spp. Esta protozoose tem como principal vetor os flebotomíneos, sendo que, no Brasil, o Lutzomyia longipalpis é a principal espécie incriminada na transmissão da leishmaniose Visceral Americana. A presença do ácido desoxirribonucleico (DNA) do parasito em ectoparasitos, como carrapatos e pulgas, tem gerado especulações quanto a existência de novos vetores no ciclo da leishmaniose. Foi objetivo deste estudo relatar a detecção molecular de Leishmania spp. em uma mutuca da espécie Tabanus importunus que parasitava um cão oligossintomático infectado por Leishmania spp. A análise molecular amplificou o DNA do protozoário na cabeça, na região torácica e no abdomen do tabanídeo, resultando como positivo para complexo Leishmania. Este é o primeiro relato da presença de DNA de Leishmania spp. em insetos dipteros da espécie T. importunus.

Cães; leishmaniose visceral canina; Mutuca; PCR; protozoário; vetores


Introduction

Leishmaniasis are an important disease, with a range of clinical and epidemiological features, more frequently reported in regions where several species of sand flies exist, particularly the species of Lutzomyia longipalpis (LAURENTI et al., 2009Laurenti MD, Silveira VM, Secundino MF, Corbett CE, Pimenta PP. Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis. Parasitol Int 2009; 58(3): 220-226. PMid:19454323. http://dx.doi.org/10.1016/j.parint.2009.05.005
http://dx.doi.org/10.1016/j.parint.2009....
; SOARES et al., 2010Soares MRA, Carvalho CC, Silva LA, Lima MSCS, Barral AMP, Rebêlo JMM, et al. Molecular analysis of natural infection of Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in Brazil. Cad Saúde Pública. 2010; 26(12): 2409-2413. PMid:21243235. http://dx.doi.org/10.1590/S0102-311X2010001200019
http://dx.doi.org/10.1590/S0102-311X2010...
; COSTA et al., 2013Costa LP, Dantas-Torres F, Da Silva FJ, Guimarães VCFV, Gaudêncio K, Brandão-Filho SP. Ecology of Lutzomyia longipalpis in an area of visceral leishmaniasis transmission in north-eastern Brazil. Acta Trop 2013; 126(2): 99-102. PMid:23369878. http://dx.doi.org/10.1016/j.actatropica.2013.01.011
http://dx.doi.org/10.1016/j.actatropica....
).

Although leishmaniasis is transmitted by sand fly bites infection via other routes have been reported, including congenital transmission in dogs (ROSYPAL et al., 2005Rosypal AC, Troy GC, Zajac AM, Frank G, Lindsay DS. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J Parasitol 2005; 91(4): 970-972. PMid:17089780. http://dx.doi.org/10.1645/GE-483R.1
http://dx.doi.org/10.1645/GE-483R.1...
; DA SILVA et al., 2009) and blood transfusions (DE FREITAS et al., 2006; GOODNOUGH, 2013Goodnough LT. Blood management: transfusion medicine comes of age. Lancet 2013; 381(9880): 1791-1792. http://dx.doi.org/10.1016/S0140-6736(13)60673-X
http://dx.doi.org/10.1016/S0140-6736(13)...
). Among other possibilities of transmission, researchers have reported the presence of DNA from this parasite in ixodids of the species Rhipicephalus sanguineus and in fleas of the species Ctenocephalides felis felis, suggesting that these arthropods may act positively in the epidemiology of this disease (OTRANTO; DANTAS-TORRES, 2010Otranto D, Dantas-Torres F. Fleas and ticks as vectors of Leishmania spp. to dogs: caution is needed. Vet Parasitol 2010; 168(1-2): 173-174. PMid:20022429. http://dx.doi.org/10.1016/j.vetpar.2009.11.016
http://dx.doi.org/10.1016/j.vetpar.2009....
). Horse flies are hematophagous insects known to be important vectors of several etiologic agents that cause diseases, particularly trypanosomiasis (SILVA et al., 1996Silva RAMS, Silva JA, Schneider RC, Freitas J, Mesquita D, Mesquita T, et al. Outbreak of trypanosomiasis due to Trypanosoma vivax (Ziemann, 1905) in bovines of the Pantanal, Brazil. Mem Inst Oswaldo Cruz 1996; 91 (5): 561-562. http://dx.doi.org/10.1590/S0074-02761996000500005
http://dx.doi.org/10.1590/S0074-02761996...
).

The aim of this paper is to report the molecular detection of Leishmania spp. in a horse fly of the species Tabanus importunus captured in the region of Andradina, state of São Paulo, Brazil.

Findings

An adult male dog of undefined breed, short-haired and predominantly black, was sent to the Veterinary Hospital of Andradina Educational College in the state of São Paulo (20.8961°, 51.37944°, 405 m altitude). This animal lived in a peri-urban zone in a house with a large yard shaded by fruit and ornamental trees, in the company of other animals such as exotic caged birds and chickens in a chicken run.

The dog was clinically characterized as oligosymptomatic for leishmaniasis (onychogryphosis, weight loss, lymphadenomegaly, muscle atrophy and dermatitis). During anamnesis at the animal's place of origin, a horse fly was observed sucking blood on the dog's left dorsal thoracic region.

In this municipality and surroundings, these tabanids frequently attack farm animals such as cattle, horses and goats, and parasitize dogs, nestlings in their nests, and even people.

The insect was captured by hand, stored in a plastic tube and frozen at −20 °C. The animal was diagnosed parasitologically as a carrier of Leishmania spp. infection, since numerous amastigote forms of Leishmania spp. were visible in the imprint of the right popliteal lymph node. The dog was euthanized according to Brazilian legislation, as per Resolution no. 1000/2012 of the Federal Council of Veterinary Medicine, and subjected to necropsy.

The dipteran collected from the animal was divided into three parts with a bistoury, and these parts were separated into aliquots and identified as follows: head (H1), thorax (T1) and abdomen (A1). When stored in the tube, the insect ejected a droplet of blood-like material from the posterior end of its abdomen (Figure 1). The tube was washed with 0.5 mL of 0.9% sterile sodium chloride solution and the sample was classified as washed (W1).

Figure 1.
Tabanus importunus infected by Leishmania spp.

The insect parts were macerated in a porcelain crucible containing 0.5 mL of the same physiological solution. Similarly, the blood sample and the lymph node fragment from the dog were collected, divided into aliquots and stored at −20 °C for analysis by polymerase chain reaction (PCR).

The aliquots were subjected to molecular analysis by PCR, using oligonucleotides that amplify the conserved region of the kinetoplast (kDNA) minicircle, using the primers 13A (5′-GTG GGG GAG GGG CGT TCT -3′) e 13B (5′-ATT TTA CAC CAA CCC CCA GTT-3′) (RODGERS et al., 1990Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as tool in the detection and diagnosis of Leishmania. Exp Parasitol 1990; 71(3): 267-275. http://dx.doi.org/10.1016/0014-4894(90)90031-7
http://dx.doi.org/10.1016/0014-4894(90)9...
). These analyses were performed by an outsourced laboratory known for its accurate diagnosis of animal and human leishmaniasis.

Part of W1 was used to prepare the smear on a microscope slide. This material was fixed and stained using a "Panótico Rápido®" kit and was examined under 1000x magnification in a light microscope equipped with a flat achromatic lens; 300 microscope fields were examined.

The molecular analysis indicated that all the samples from the arthropod (H1, T1, A1 and W1) and from the dog were positive for Leishmania complex, and the reference value adopted was the negative result. Microscopic analysis of the smear from W1 showed amastigotes forms of Leishmania spp. (Figure 1).

Leishmaniasis is an important zoonosis whose cycle is classically characterized by the presence of dipterous vectors of the genus Lutzomyia (GALATI et al., 2003Galati EAB, Nunes VLB, Cristaldo G, Da Rocha HC. Aspectos do comportamento da fauna flebotomínea (Diptera: Psychodidae) em foco de leishmaniose visceral e tegumentar na Serra da Bodoquena e area adjacente, Estado de Mato Grosso do Sul, Brasil. Rev Patol Trop 2003; 32(2): 235-261.; KAMHAWI, 2006Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol 2006; 22(9): 439-445 PMid:16843727. http://dx.doi.org/10.1016/j.pt.2006.06.012
http://dx.doi.org/10.1016/j.pt.2006.06.0...
; SILVA et al., 2008Silva EA, Andreotti R, Dias ES, Barros JC, Brazuna JC. Detection of Leishmania DNA in phlebotomines captured in Campo Grande, Mato Grosso do Sul, Brazil. Exp Parasitol 2008; 119(3): 343-348. PMid:18456262. http://dx.doi.org/10.1016/j.exppara.2008.03.011
http://dx.doi.org/10.1016/j.exppara.2008...
). However, entomological studies carried out in different Brazilian municipalities did not confirm the presence of this phlebotomine in areas endemic to L. chagasi (DANTAS-TORRES, 2006Dantas-Torres F. Do any insects other than phlebotomine sand flies (Diptera: Psychodidae) transmit Leishmania infantum (Kinetoplastida: Trypanosomatidae) from dog to dog? Vet Parasitol 2006; 136(3-4): 379-380. PMid:16417969. http://dx.doi.org/10.1016/j.vetpar.2005.12.010
http://dx.doi.org/10.1016/j.vetpar.2005....
). Thus, the possible existence of a new vector has been suggested in the epidemiology of this parasitosis (PAZ, 2010a).

Several researchers have mentioned the possibility that R. sanguineus acts as a vector of leishmaniasis (PAZ et al., 2010bPaz GF, Ribeiro MFB, Michlasky EM, Da Rocha Lima ACVM, França-Silva JC, et al. Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 2010b; 106(2): 523-528. PMid:20012095. http://dx.doi.org/10.1007/s00436-009-1697-1
http://dx.doi.org/10.1007/s00436-009-169...
), either through its bite (DANTAS-TORRES et al., 2010Dantas-Torres F, Lorusso V, Testini G, DE Paiva-Cavalcanti M, Figueredo LA, Stanneck D, et al. Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 2010; 106(4): 857-860. PMid:20127362. http://dx.doi.org/10.1007/s00436-010-1722-4
http://dx.doi.org/10.1007/s00436-010-172...
) or through the ingestion of infected ticks (COUTINHO et al., 2005Coutinho MTZ, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, et al. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol 2005; 128(1-2): 149-155. PMid:15725545. http://dx.doi.org/10.1016/j.vetpar.2004.11.011
http://dx.doi.org/10.1016/j.vetpar.2004....
). Similarly, fleas have been reported as vectors of Leishmania spp. (COUTINHO; LINARDI, 2007Coutinho MTZ, Linardi PM. Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Vet Parasitol 2007; 147(3-4): 320-325. PMid:17521814. http://dx.doi.org/10.1016/j.vetpar.2007.04.008
http://dx.doi.org/10.1016/j.vetpar.2007....
; PAZ, 2010a). However, the detection of Leishmania spp. DNA does not suffice to imply a species as vector (SAVANI et al., 2009Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, Zampieri RA, Floeter-Winter LM. The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil. Vet Parasitol 2009; 160(1-2): 18-24. PMid:19062193. http://dx.doi.org/10.1016/j.vetpar.2008.10.090
http://dx.doi.org/10.1016/j.vetpar.2008....
).

In this sense, coinfection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi has already been found in a dog from the state of Mato Grosso do Sul (SAVANI et al., 2005Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, De Araujo FS, Ilha IMN, et al. Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz 2005; 100(7): 739-741. http://dx.doi.org/10.1590/S0074-02762005000700011
http://dx.doi.org/10.1590/S0074-02762005...
).

In this region, the non-selective behavior of these tabanids for host species is worthy of note, as it is an endemic area for human and canine visceral leishmaniasis. This fact is worth highlighting because tabanids are considered important vectors of trypanosomiasis in animals (NUNES, 1996; FRANKE et al., 1994Franke CR, Greiner M, Mehlitz D. Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Poconé (Mato Grosso, Brazil). Acta Trop 1994; 58(2): 159-169. http://dx.doi.org/10.1016/0001-706X(94)90055-8
http://dx.doi.org/10.1016/0001-706X(94)9...
; HERRERA et al., 2004Herrera HM, Dávila AMR, Norek A, Abreu UG, Souza SS, D'Andrea PS, et al. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol 2004; 125(3-4): 263-275. PMid:15482883. http://dx.doi.org/10.1016/j.vetpar.2004.07.013
http://dx.doi.org/10.1016/j.vetpar.2004....
).

The epidemiology of leishmaniasis is a constantly expanding theme and the action of new vectors in the cycle of this disease must be considered. Thus, this paper offers the first report of the presence of amastigotes forms and DNA of Leishmania spp. in a horse fly of the species T. importunus parasitizing a dog that was a carrier of canine visceral leishmaniasis, suggesting the possibility that the dipteran of species Tabanus importunus act as mechanical vectors in the cycle of this zoonosis.

References

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    » http://dx.doi.org/10.1016/j.actatropica.2013.01.011
  • Coutinho MTZ, Linardi PM. Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Vet Parasitol 2007; 147(3-4): 320-325. PMid:17521814. http://dx.doi.org/10.1016/j.vetpar.2007.04.008
    » http://dx.doi.org/10.1016/j.vetpar.2007.04.008
  • Coutinho MTZ, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, et al. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol 2005; 128(1-2): 149-155. PMid:15725545. http://dx.doi.org/10.1016/j.vetpar.2004.11.011
    » http://dx.doi.org/10.1016/j.vetpar.2004.11.011
  • Dantas-Torres F, Lorusso V, Testini G, DE Paiva-Cavalcanti M, Figueredo LA, Stanneck D, et al. Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 2010; 106(4): 857-860. PMid:20127362. http://dx.doi.org/10.1007/s00436-010-1722-4
    » http://dx.doi.org/10.1007/s00436-010-1722-4
  • Dantas-Torres F. Do any insects other than phlebotomine sand flies (Diptera: Psychodidae) transmit Leishmania infantum (Kinetoplastida: Trypanosomatidae) from dog to dog? Vet Parasitol 2006; 136(3-4): 379-380. PMid:16417969. http://dx.doi.org/10.1016/j.vetpar.2005.12.010
    » http://dx.doi.org/10.1016/j.vetpar.2005.12.010
  • Da Silva SM, Ribeiro VM, Ribeiro RR, Tafuri WL, Melo MN, Michalick MSM. First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil. Vet Parasitol 2009; 166(1-2): 159-162. PMid:19733439. http://dx.doi.org/10.1016/j.vetpar.2009.08.011
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  • Franke CR, Greiner M, Mehlitz D. Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Poconé (Mato Grosso, Brazil). Acta Trop 1994; 58(2): 159-169. http://dx.doi.org/10.1016/0001-706X(94)90055-8
    » http://dx.doi.org/10.1016/0001-706X(94)90055-8
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  • Goodnough LT. Blood management: transfusion medicine comes of age. Lancet 2013; 381(9880): 1791-1792. http://dx.doi.org/10.1016/S0140-6736(13)60673-X
    » http://dx.doi.org/10.1016/S0140-6736(13)60673-X
  • Herrera HM, Dávila AMR, Norek A, Abreu UG, Souza SS, D'Andrea PS, et al. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol 2004; 125(3-4): 263-275. PMid:15482883. http://dx.doi.org/10.1016/j.vetpar.2004.07.013
    » http://dx.doi.org/10.1016/j.vetpar.2004.07.013
  • Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol 2006; 22(9): 439-445 PMid:16843727. http://dx.doi.org/10.1016/j.pt.2006.06.012
    » http://dx.doi.org/10.1016/j.pt.2006.06.012
  • Laurenti MD, Silveira VM, Secundino MF, Corbett CE, Pimenta PP. Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis. Parasitol Int 2009; 58(3): 220-226. PMid:19454323. http://dx.doi.org/10.1016/j.parint.2009.05.005
    » http://dx.doi.org/10.1016/j.parint.2009.05.005
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    » http://dx.doi.org/10.1016/j.vetpar.2009.11.016
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  • Paz GF, Ribeiro MFB, Michlasky EM, Da Rocha Lima ACVM, França-Silva JC, et al. Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 2010b; 106(2): 523-528. PMid:20012095. http://dx.doi.org/10.1007/s00436-009-1697-1
    » http://dx.doi.org/10.1007/s00436-009-1697-1
  • Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as tool in the detection and diagnosis of Leishmania. Exp Parasitol 1990; 71(3): 267-275. http://dx.doi.org/10.1016/0014-4894(90)90031-7
    » http://dx.doi.org/10.1016/0014-4894(90)90031-7
  • Rosypal AC, Troy GC, Zajac AM, Frank G, Lindsay DS. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J Parasitol 2005; 91(4): 970-972. PMid:17089780. http://dx.doi.org/10.1645/GE-483R.1
    » http://dx.doi.org/10.1645/GE-483R.1
  • Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, Zampieri RA, Floeter-Winter LM. The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil. Vet Parasitol 2009; 160(1-2): 18-24. PMid:19062193. http://dx.doi.org/10.1016/j.vetpar.2008.10.090
    » http://dx.doi.org/10.1016/j.vetpar.2008.10.090
  • Savani ESMM, Nunes VLB, Galati EAB, Castilho TM, De Araujo FS, Ilha IMN, et al. Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz 2005; 100(7): 739-741. http://dx.doi.org/10.1590/S0074-02762005000700011
    » http://dx.doi.org/10.1590/S0074-02762005000700011
  • Silva EA, Andreotti R, Dias ES, Barros JC, Brazuna JC. Detection of Leishmania DNA in phlebotomines captured in Campo Grande, Mato Grosso do Sul, Brazil. Exp Parasitol 2008; 119(3): 343-348. PMid:18456262. http://dx.doi.org/10.1016/j.exppara.2008.03.011
    » http://dx.doi.org/10.1016/j.exppara.2008.03.011
  • Silva RAMS, Silva JA, Schneider RC, Freitas J, Mesquita D, Mesquita T, et al. Outbreak of trypanosomiasis due to Trypanosoma vivax (Ziemann, 1905) in bovines of the Pantanal, Brazil. Mem Inst Oswaldo Cruz 1996; 91 (5): 561-562. http://dx.doi.org/10.1590/S0074-02761996000500005
    » http://dx.doi.org/10.1590/S0074-02761996000500005
  • Soares MRA, Carvalho CC, Silva LA, Lima MSCS, Barral AMP, Rebêlo JMM, et al. Molecular analysis of natural infection of Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in Brazil. Cad Saúde Pública. 2010; 26(12): 2409-2413. PMid:21243235. http://dx.doi.org/10.1590/S0102-311X2010001200019
    » http://dx.doi.org/10.1590/S0102-311X2010001200019

Publication Dates

  • Publication in this collection
    Oct-Dec 2013

History

  • Received
    29 Apr 2013
  • Accepted
    4 June 2013
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
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