Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

Marize Quinhones Pires Maria de Fátima Madeira Vânia Rita Elias Pinheiro Bittencourt Raquel da Silva Pacheco About the authors

Abstract

Introduction

During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi.

Methods

Specific polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) assays were performed.

Results

PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments.

Conclusions

This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Visceral leishmaniasis; Tegumentary leishmaniasis; Restriction fragment length polymorphism-polymerase chain reaction


Visceral leishmaniasis (VL) and tegumentary leishmaniasis (TL) are zoonoses of great importance for public health. In the State of Rio de Janeiro, Leishmania (Viannia) braziliensis is the most prevalent species implicated in the epidemiological cycle of TL. Its transmission occurs in periurban areas in which primitive rain forest vegetation is being depredated due to disorderly human occupation11. Marzochi MCA, Marzochi KBF. Tegumentary and visceral leishmaniases in Brazil: emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 1994; 10 (Suppl II):359-375., and infections in man, dogs and horses have been reported22. Marzochi MCA, Coutinho SG, Souza WJ, Toledo LM, Grimaldi Jr G, Momen H, et al. Canine visceral leishmaniasis in Rio de Janeiro, Brazil. Clinical, parasitological, therapeutical and epidemiological findings (1977-1983). Mem Inst Oswaldo Cruz 1985; 80:349-357.,33. Barbosa-Santos EGO, Marzochi MCA, Urtado W, Queirós F, Chicarino J, Pacheco RS. Leishmaniasis disseminated by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays. Mem Inst Oswaldo Cruz 1994; 89:217-220.. Canine VL, which is caused by Leishmania (Leishmania) chagasi, is endemic in the Municipality of Rio de Janeiro. Dogs represent one of the main reservoirs in urban areas in which the disease has been observed due to several factors that influence the natural epidemiological scenario44. Ministério da Saúde: Manual de vigilância e controle da leishmaniose visceral. Brasília: Editora MS; 2006.. The overlapping transmission of TL caused by L. (V.) braziliensis and VL caused by L. (L.) chagasi has been reported in certain areas of the Municipality of Rio de Janeiro. Mixed infection with both parasites has already been reported in a patient55. Oliveira-Neto MP, Marzochi MCA, Grimaldi Jr G, Pacheco RS, Toledo LM, Momen H. Concurrent human infection with Leishmania donovani chagasi and Leishmania braziliensis braziliensis. Ann Trop Med Parasitol 1986; 80:587-592. and in a dog66. Madeira MF, Schubach A, Schubach TPM, Pacheco RS, Oliveira FS, Pereira SA, et al. Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro. Trans R Soc Trop Med Hyg 2006; 100:442-445.. Fortunately, according to Marzochi et al.77. Marzochi MCA, Fagundes A, Andrade MV, Souza MB, Madeira MF, Mouta-Confort E, et al. Visceral leishmaniasis in Rio de Janeiro, Brazil: eco-epidemiological aspects and control. Rev Soc Bras Med Trop 2009; 42:570-580., no new human cases of co-infection have been registered since, but there is concern about the persistence of canine seroprevalence. Control measures are based on interrupting the transmission cycle, which involves the diagnosis and treatment of human cases and vector control through insecticides and serological screening, with the subsequent culling of dogs found to be seropositive44. Ministério da Saúde: Manual de vigilância e controle da leishmaniose visceral. Brasília: Editora MS; 2006..

The present article discusses the detection of mixed TL and VL infections in two of seventeen dogs from endemic areas of Rio de Janeiro, Brazil, which tested seropositive by indirect immunofluorescence (IIF) analysis of serum samples. The occurrence of canine cases with both diseases in the same geographic area impairs the diagnosis and implementation of control measures.

The animals included in this study were referred to the Zoonosis Service of the Instituto de Pesquisa Clínica Evandro Chagas-Fundação Oswaldo Cruz (IPEC-FIOCRUZ) with an indication for euthanasia according to the recommendations of the Brazilian Program for the Control of Leishmaniasis44. Ministério da Saúde: Manual de vigilância e controle da leishmaniose visceral. Brasília: Editora MS; 2006. after serological tests by IIF on serum samples, which were performed by the Epidemiology Service of the Municipality of Rio de Janeiro. This study was approved by the Ethics Committee on Animal Experimentation of the Fundação Oswaldo Cruz (CEUA/FIOCRUZ; program n° L-023/06).

All studied dogs were from urban and periurban areas of the Municipality of Rio de Janeiro and presented IIF titers ranging from 1:80 to 1:1,280. In certain animals, clinical symptoms of VL were evident, whereas others were asymptomatic. Cutaneous lesions were frequent (Table 1).

TABLE 1 -
The serological titers, clinical status and PCR results of the 17 dogs from Rio de Janeiro.

Fragments of the cutaneous lesions, intact skin from the scapular region, cervical lymph nodes and spleen from all animals were collected after thiopental-overdose euthanasia and submitted for polymerase chain reaction (PCR) analyses using primers for the variable regions of kinetoplast DNA (kDNA) minicircles. Specific primers for the L. braziliensis complex (5′-GGGGTTGGTGTAATATAGTGG-3′ and 5′-CTAATTGTGCACGGGGAGG-3′)88. De Bruijn MH, Barker DC. Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Trop 1992; 52:45-58. and for the Leishmania donovani complex (5′-CCAGTTTCCCGCCCCG-3′ and 5′-GGGGTTGGTGTAAAATAG-3′)99. Smyth AJ, Ghosh A, Hassan MQ, Basu D, De Bruijn MH, Mallik KK, et al. Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of Kala-azar patients. Parasitol 1992; 105:183-192. were adopted, as previously described1010. Oliveira FS, Pirmez C, Pires MQ, Brazil RP, Pacheco RS. PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil. Vet Parasitol 2005; 129:219-227.,1111. Silva ES, Gontijo CMF, Pacheco RS, Brazil RP. Diagnosis of human visceral leishmaniasis by PCR using blood samples on filter paper. Genet Mol Res 2004; 3:251-257.. Then, the amplified PCR products were visualized on agarose gels. Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) analyses were performed with a panel of four restriction enzymes (Msp I, Rsa I, Hinf I and Mbo I) to confirm the specificity of the amplified kDNA minicircle products.

All of the canine biopsy fragments, except for two from cutaneous lesions, produced the expected 800bp diagnostic bands after PCR with primers D1/D2. Those fragments of cutaneous lesions that tested negative were submitted to PCR assays with the primers B1/B2, and the expected 750bp diagnostic bands were observed (Figure 1A).

FIGURE 1 -
A: Specific PCR products in 1.5% agarose gel: line M: DNA ladder, 100bp; primers B1/B2: line 1: dog A43, cutaneous lesion; line 2: dog A63, cutaneous lesion; line 3: Leishmania (Viannia) braziliensis reference strain; lines 4 and 10: negative controls; primers D1/D2: lines 5-6: dog A43, spleen fragment and cervical lymph node, respectively; lines 7-8: dog A63, spleen fragment and cervical lymph node, respectively; line 9: Leishmania (Leishmania) chagasi reference strain. B: RFLP analyses in high-resolution 1.8% agarose gel after digestion with the enzymes Rsa I and Hinf I: lines 1 and 4: dog A43, cutaneous lesion; lines 2 and 5: dog A63, cutaneous lesion; lines 3 and 6: Leishmania (Viannia) braziliensis reference strain. C and D: RFLP analyses with Rsa I and Mbo I: lines 1-2: dog A43, spleen and cervical lymph node, respectively; lines 3-4: dog A63, spleen and cervical lymph node, respectively; line 5: Leishmania (Leishmania) chagasi reference strain. E: RFLP analyses with Msp I: line 1: dog A43, cutaneous lesion; line 2: dog A63, cutaneous lesion; line 3: Leishmania (Viannia) braziliensis reference strain; lines 4-5: dog A43, spleen and cervical lymph node, respectively; line 6-7: dog A63, spleen and cervical lymph node, respectively; line 8: Leishmania (Leishmania) chagasi reference strain. PCR: polymerase chain reaction; DNA: deoxyribonucleic acid; RFLP: restriction fragment length polymorphisms.

PCR was performed in combination with RFLP-PCR to confirm the presence of L. (V.) braziliensis deoxyribonucleic acid (DNA) in the cutaneous lesion biopsies and L. (L.) chagasi DNA in the spleen and lymph node fragments from two dogs (dogs A43 and A63, Table 1). In Figures 1B, 1C, 1D and 1E, the PCR/RFLP results confirming the mixed infection are observed. Because previous results from our group1212. Lopes UG, Momen H, Grimaldi Jr G, Marzochi MCA, Pacheco RS, Morel CM. Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis. J Parasitol 1984; 70:89-98.,1313. Pacheco RS, Lopes UG, Morel CM, Grimaldi Jr G, Momen H. Schizodeme analysis of Leishmania isolates and comparison with some phenotypic techniques. In: Rioux JA, editor. Leishmania, Taxonomie et Phylogenése. Applications éco-épidémiologiques. Montpellier: IMEEE; 1986. p.57-65. have demonstrated that the restriction enzymes Msp I, Rsa I, Hinf I and Mbo I are the most appropriate for typing Leishmania species from the subgenera Viannia and Leishmania, these enzymes were adopted in the present study. The Msp I restriction enzyme linearizes the kDNA minicircles, displaying a major band of approximately 750bp in the case of Leishmania (V.) species. In contrast, a polymorphic restriction profile is always observed in L (L.) chagasi1212. Lopes UG, Momen H, Grimaldi Jr G, Marzochi MCA, Pacheco RS, Morel CM. Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis. J Parasitol 1984; 70:89-98..

Herein, the Hinf I restriction patterns of the amplified parasitic DNA from cutaneous lesions were similar to the patterns of the L. (V.) braziliensis (MHOM/BR/75/M2903) reference strain kDNA. The Rsa I restriction patterns obtained with the amplified products from the spleen and lymph node fragments were also similar compared with the patterns of the L. (L.) chagasi (MHOM/BR/74/PP75) reference strain kDNA (Figure 1C). Both subgenera were confirmed after digestion with Msp I (Figure 1E). RFLP-PCR analysis with the restriction enzymes Msp I, Rsa I and Mbo I corroborated the PCR results, justifying the use of such a technique in Leishmania species identification.

In clinical-epidemiological surveillance, emphasizing the need to perform extensive sampling in dogs with cutaneous lesions is important, particularly in endemic areas in which both diseases overlap, such as certain rural areas in Rio de Janeiro. In these areas, where closely related etiological groups are present, the interpretation of serological data is a limiting aspect due to possible serological cross-reactions. Although Trypanosoma cruzi infection is unknown in the Municipality of Rio de Janeiro, a new species, Trypanosoma caninum, was recently described in dogs1414. Barros JH, Almeida AB, Figueiredo FB, Sousa VR, Fagundes A, Pinto AG, et al. Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control? Trans R Soc Trop Med Hyg 2012; 106:419-423.. In this scenario, a reassessment of control measures is required.

The control of leishmaniasis is relatively complex, particularly in areas where both the tegumentary and visceral forms of the disease co-exist. As a control measure, the Brazilian government usually culls seropositive dogs44. Ministério da Saúde: Manual de vigilância e controle da leishmaniose visceral. Brasília: Editora MS; 2006.. However, according to a recent review1515. Costa CHN. How effective is dog culling in controlling zoonotic visceral leishmaniasis? A critical evaluation of the science, politics and ethics behind this public health policy. Rev Soc Bras Med Trop 2011; 44:232-242., the strategy of killing dogs is hampered for several reasons, including the low accuracy of the methods used to assess the infectivity of dogs and the high replacement rate of these animals. In this scenario, a search for sensitive and specific molecular tools is needed to distinguish dogs infected with L. (V.) braziliensis, thus preventing unnecessary sacrifice. The results presented here show the usefulness of specific PCR assays and the RFLP technique for differentiating between L. (V.) braziliensis and L. (L.) chagasi and may contribute to providing support for control programs. Conversely, dogs with TL and VL co-infection would be subjected to euthanasia according to the guidelines of the Ministry of Health. Concerning control measures, including the detection and treatment of human cases, the disposal of dogs with VL, the efforts to control vectors with systematic indoor and outdoor spraying and the use of collars and mosquito nets impregnated with insecticides, the latter measure alone would likely be more efficient than the first two measures together. Finally, the development of human vaccines should also be considered as a high priority.

REFERENCES

  • 1
    Marzochi MCA, Marzochi KBF. Tegumentary and visceral leishmaniases in Brazil: emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 1994; 10 (Suppl II):359-375.
  • 2
    Marzochi MCA, Coutinho SG, Souza WJ, Toledo LM, Grimaldi Jr G, Momen H, et al. Canine visceral leishmaniasis in Rio de Janeiro, Brazil. Clinical, parasitological, therapeutical and epidemiological findings (1977-1983). Mem Inst Oswaldo Cruz 1985; 80:349-357.
  • 3
    Barbosa-Santos EGO, Marzochi MCA, Urtado W, Queirós F, Chicarino J, Pacheco RS. Leishmaniasis disseminated by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays. Mem Inst Oswaldo Cruz 1994; 89:217-220.
  • 4
    Ministério da Saúde: Manual de vigilância e controle da leishmaniose visceral. Brasília: Editora MS; 2006.
  • 5
    Oliveira-Neto MP, Marzochi MCA, Grimaldi Jr G, Pacheco RS, Toledo LM, Momen H. Concurrent human infection with Leishmania donovani chagasi and Leishmania braziliensis braziliensis. Ann Trop Med Parasitol 1986; 80:587-592.
  • 6
    Madeira MF, Schubach A, Schubach TPM, Pacheco RS, Oliveira FS, Pereira SA, et al. Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro. Trans R Soc Trop Med Hyg 2006; 100:442-445.
  • 7
    Marzochi MCA, Fagundes A, Andrade MV, Souza MB, Madeira MF, Mouta-Confort E, et al. Visceral leishmaniasis in Rio de Janeiro, Brazil: eco-epidemiological aspects and control. Rev Soc Bras Med Trop 2009; 42:570-580.
  • 8
    De Bruijn MH, Barker DC. Diagnosis of New World leishmaniasis: specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Trop 1992; 52:45-58.
  • 9
    Smyth AJ, Ghosh A, Hassan MQ, Basu D, De Bruijn MH, Mallik KK, et al. Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of Kala-azar patients. Parasitol 1992; 105:183-192.
  • 10
    Oliveira FS, Pirmez C, Pires MQ, Brazil RP, Pacheco RS. PCR-based diagnosis for detection of Leishmania in skin and blood of rodents from an endemic area of cutaneous and visceral leishmaniasis in Brazil. Vet Parasitol 2005; 129:219-227.
  • 11
    Silva ES, Gontijo CMF, Pacheco RS, Brazil RP. Diagnosis of human visceral leishmaniasis by PCR using blood samples on filter paper. Genet Mol Res 2004; 3:251-257.
  • 12
    Lopes UG, Momen H, Grimaldi Jr G, Marzochi MCA, Pacheco RS, Morel CM. Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis. J Parasitol 1984; 70:89-98.
  • 13
    Pacheco RS, Lopes UG, Morel CM, Grimaldi Jr G, Momen H. Schizodeme analysis of Leishmania isolates and comparison with some phenotypic techniques. In: Rioux JA, editor. Leishmania, Taxonomie et Phylogenése. Applications éco-épidémiologiques. Montpellier: IMEEE; 1986. p.57-65.
  • 14
    Barros JH, Almeida AB, Figueiredo FB, Sousa VR, Fagundes A, Pinto AG, et al. Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control? Trans R Soc Trop Med Hyg 2012; 106:419-423.
  • 15
    Costa CHN. How effective is dog culling in controlling zoonotic visceral leishmaniasis? A critical evaluation of the science, politics and ethics behind this public health policy. Rev Soc Bras Med Trop 2011; 44:232-242.

  • FINANCIAL SUPPORT This work received fi nancial support from Conselho Nacional de Desenvolvimento Científi co e Tecnológico (CNPq), Fundação Carlos Chagas de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Kinder do Brasil (IKB). RS Pacheco is a Brazilian CNPq investigator.

Publication Dates

  • Publication in this collection
    Mar-Apr 2014

History

  • Received
    29 Jan 2013
  • Accepted
    25 Mar 2013
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