Table of Contents
Advances in Pharmaceutics
Volume 2015 (2015), Article ID 160208, 13 pages
http://dx.doi.org/10.1155/2015/160208
Research Article

Unresponsiveness of Experimental Canine Leishmaniosis to a New Amphotericin B Formulation

1Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro s/n, 28040 Madrid, Spain
2Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Plaza Ramon y Cajal, 28040 Madrid, Spain
3Farmacia y Tecnologia Farmaceutica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramon y Cajal, 28040 Madrid, Spain
4Department of Veterinary Medicine, University of Bari, Valenzano, 70010 Bari, Italy

Received 10 September 2014; Accepted 7 December 2014

Academic Editor: Maria J. Morilla

Copyright © 2015 Leticia Hernández et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Linked References

  1. G. Baneth, A. F. Koutinas, L. Solano-Gallego, P. Bourdeau, and L. Ferrer, “Canine leishmaniosis—new concepts and insights on an expanding zoonosis: part one,” Trends in Parasitology, vol. 24, no. 7, pp. 324–330, 2008. View at Publisher · View at Google Scholar · View at Scopus
  2. M. Gramiccia and L. Gradoni, “The current status of zoonotic leishmaniases and approaches to disease control,” International Journal for Parasitology, vol. 35, no. 11-12, pp. 1169–1180, 2005. View at Publisher · View at Google Scholar · View at Scopus
  3. R. Killick-Kendrick, “Phlebotomine vectors of the leishmaniases: a review,” Medical and Veterinary Entomology, vol. 4, no. 1, pp. 1–24, 1990. View at Publisher · View at Google Scholar · View at Scopus
  4. R. J. Slappendel, “Canine leishmaniasis. A review based on 95 cases in The Netherlands,” Veterinary Quarterly, vol. 10, no. 1, pp. 1–16, 1988. View at Publisher · View at Google Scholar · View at Scopus
  5. L. Solano-Gallego, G. Mirá, A. Koutinas et al., “LeishVet guidelines for the practical management of canine leishmaniosis,” Parasites and Vectors, vol. 4, article 86, 2011. View at Publisher · View at Google Scholar · View at Scopus
  6. A. F. Koutinas, Z. S. Polizopoulou, M. N. Saridomichelakis, D. Argyriadis, A. Fytianou, and K. G. Plevraki, “Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases (1989–1996),” Journal of the American Animal Hospital Association, vol. 35, no. 5, pp. 376–383, 1999. View at Publisher · View at Google Scholar · View at Scopus
  7. M. N. Saridomichelakis, “Advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications,” Veterinary Dermatology, vol. 20, no. 5-6, pp. 471–489, 2009. View at Publisher · View at Google Scholar · View at Scopus
  8. F. A. L. Costa, H. Goto, L. C. B. Saldanha et al., “Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis,” Veterinary Pathology, vol. 40, no. 6, pp. 677–684, 2003. View at Publisher · View at Google Scholar · View at Scopus
  9. C. Noli and S. T. Auxilia, “Treatment of canine old world visceral leishmaniasis: a systematic review,” Veterinary Dermatology, vol. 16, no. 4, pp. 213–232, 2005. View at Publisher · View at Google Scholar · View at Scopus
  10. L. Manna, S. Reale, F. Vitale, E. Picillo, L. M. Pavone, and A. E. Gravino, “Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol,” Veterinary Journal, vol. 177, no. 2, pp. 279–282, 2008. View at Publisher · View at Google Scholar · View at Scopus
  11. F. A. Ikeda-Garcia, R. S. Lopes, F. J. Marques et al., “Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate,” Veterinary Parasitology, vol. 143, no. 3-4, pp. 254–259, 2007. View at Publisher · View at Google Scholar · View at Scopus
  12. R. R. Ribeiro, E. P. Moura, V. M. Pimentel et al., “Reduced tissue parasitic load and infectivity to sand flies in dogs naturally infected by Leishmania (Leishmania) chagasi following treatment with a liposome formulation of meglumine antimoniate,” Antimicrobial Agents and Chemotherapy, vol. 52, no. 7, pp. 2564–2572, 2008. View at Publisher · View at Google Scholar · View at Scopus
  13. G. Miró, R. Gálvez, C. Fraile, M. A. Descalzo, and R. Molina, “Infectivity to Phlebotomus perniciosus of dogs naturally parasitized with Leishmania infantum after different treatments,” Parasites and Vectors, vol. 4, article 52, 2011. View at Publisher · View at Google Scholar · View at Scopus
  14. P. Denerolle and G. Bourdoiseau, “Combination allopurinol and antimony treatment versus antimony alone and allopurinol alone in the treatment of canine leishmaniasis (96 cases),” Journal of Veterinary Internal Medicine, vol. 13, no. 5, pp. 413–415, 1999. View at Publisher · View at Google Scholar · View at Scopus
  15. G. Miró, G. Oliva, I. Cruz et al., “Multicentric, controlled clinical study to evaluate effectiveness and safety of miltefosine and allopurinol for canine leishmaniosis,” Veterinary Dermatology, vol. 20, no. 5-6, pp. 397–404, 2009. View at Publisher · View at Google Scholar · View at Scopus
  16. M. Mateo, L. Maynard, C. Vischer, P. Bianciardi, and G. Miró, “Comparative study on the short term efficacy and adverse effects of miltefosine and meglumine antimoniate in dogs with natural leishmaniosis,” Parasitology Research, vol. 105, no. 1, pp. 155–162, 2009. View at Publisher · View at Google Scholar · View at Scopus
  17. J. Lamothe, “Activity of amphotericin B in lipid emulsion in the initial treatment of canine leishmaniasis,” Journal of Small Animal Practice, vol. 42, no. 4, pp. 170–175, 2001. View at Publisher · View at Google Scholar · View at Scopus
  18. G. Oliva, L. Gradoni, P. Ciaramella et al., “Activity of liposomal amphotericin B (AmBisome) in dogs naturally infected with Leishmania infantum,” Journal of Antimicrobial Chemotherapy, vol. 36, no. 6, pp. 1013–1019, 1995. View at Publisher · View at Google Scholar · View at Scopus
  19. G. Oliva, L. Gradoni, L. Cortese et al., “Comparative efficacy of meglumine antimoniate and aminosidine sulphate, alone or in combination, in canine leishmaniasis,” Annals of Tropical Medicine and Parasitology, vol. 92, no. 2, pp. 165–171, 1998. View at Publisher · View at Google Scholar · View at Scopus
  20. J. A. Vexenat, P. L. Olliaro, J. A. F. de Castro et al., “Clinical recovery and limited cure in canine visceral leishmaniasis treated with aminosidine (paromomycin),” The American Journal of Tropical Medicine and Hygiene, vol. 58, no. 4, pp. 448–453, 1998. View at Google Scholar · View at Scopus
  21. A. Rhalem, H. Sahibi, N. Guessous-Idrissi et al., “Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum,” Veterinary Parasitology, vol. 81, no. 3, pp. 173–184, 1999. View at Publisher · View at Google Scholar · View at Scopus
  22. M. G. Pennisi, M. de Majo, M. Masucci, D. Britti, F. Vitale, and R. Del Maso, “Efficacy of the treatment of dogs with leishmaniosis with a combination of metronidazole and spiramycin,” Veterinary Record, vol. 156, no. 11, pp. 346–349, 2005. View at Publisher · View at Google Scholar · View at Scopus
  23. P. Bianciardi, A. Fasanella, V. F. Manzillo et al., “The efficacy of enrofloxacin, alone or combined with metronidazole, in the therapy of canine leishmaniasis,” Parasitology Research, vol. 93, no. 6, pp. 486–492, 2004. View at Publisher · View at Google Scholar · View at Scopus
  24. S. Rougier, I. Vouldoukis, S. Fournel, S. Pérès, and F. Woehrlé, “Efficacy of different treatment regimens of marbofloxacin in canine visceral leishmaniosis: a pilot study,” Veterinary Parasitology, vol. 153, no. 3-4, pp. 244–254, 2008. View at Publisher · View at Google Scholar · View at Scopus
  25. L. Hernández, R. Gálvez, A. Montoya et al., “First study on efficacy and tolerability of a new alkylphosphocholine molecule (oleylphosphocholine—OlPC) in the treatment of canine leishmaniosis due to Leishmania infantum,” Parasitology Research, vol. 113, no. 1, pp. 157–164, 2014. View at Publisher · View at Google Scholar · View at Scopus
  26. P. Gómez-Ochoa, J. A. Castillo, M. Gascón, J. J. Zarate, F. Alvarez, and C. G. Couto, “Use of domperidone in the treatment of canine visceral Leishmaniasis: a clinical trial,” Veterinary Journal, vol. 179, no. 2, pp. 259–263, 2009. View at Publisher · View at Google Scholar · View at Scopus
  27. J. D. Dutcher, M. B. Young, J. H. Sherman, W. Hibbits, and D. R. Walters, “Chemical studies on amphotericin B. I. Preparation of the hydrogenation product and isolation of mycosamine, an acetolysis product,” Antibiotics Annual, pp. 866–869, 1956. View at Google Scholar
  28. G. Baneth and S. E. Shaw, “Chemotherapy of canine leishmaniosis,” Veterinary Parasitology, vol. 106, no. 4, pp. 315–324, 2002. View at Publisher · View at Google Scholar · View at Scopus
  29. S. C. Kinsky, “Antibiotic interaction with model membranes,” Annual Review of Pharmacology, vol. 10, pp. 119–142, 1970. View at Publisher · View at Google Scholar · View at Scopus
  30. F. Chappuis, S. Sundar, A. Hailu et al., “Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?” Nature Reviews Microbiology, vol. 5, no. 11, pp. 873–882, 2007. View at Publisher · View at Google Scholar · View at Scopus
  31. S. Sundar, P. K. Sinha, M. Rai et al., “Comparison of short-course multidrug treatment with standard therapy for visceral leishmaniasis in India: An open-label, non-inferiority, randomised controlled trial,” The Lancet, vol. 377, no. 9764, pp. 477–486, 2011. View at Publisher · View at Google Scholar · View at Scopus
  32. J. D. Berman, G. Ksionski, W. L. Chapman, V. B. Waits, and W. L. Hanson, “Activity of amphotericin B cholesterol dispersion (Amphocil) in experimental visceral leishmaniasis,” Antimicrobial Agents and Chemotherapy, vol. 36, no. 9, pp. 1978–1980, 1992. View at Publisher · View at Google Scholar · View at Scopus
  33. R. Laniado-Laborín and M. N. Cabrales-Vargas, “Amphotericin B: side effects and toxicity,” Revista Iberoamericana de Micologia, vol. 26, no. 4, pp. 223–227, 2009. View at Publisher · View at Google Scholar · View at Scopus
  34. P. Ciaramella and M. Corona, “Canine leishmaniasis: therapeutic aspects,” Compendium of Continuing Education for the Practicing Veterinarian, vol. 25, pp. 370–375, 2003. View at Google Scholar
  35. P. M. Daftarian, G. W. Stone, L. Kovalski et al., “A targeted and adjuvanted nanocarrier lowers the effective dose of liposomal amphotericin B and enhances adaptive immunity in murine cutaneous leishmaniasis,” Journal of Infectious Diseases, vol. 208, no. 11, pp. 1914–1922, 2013. View at Publisher · View at Google Scholar · View at Scopus
  36. J. A. Sánchez-Brunete, M. A. Dea, S. Rama et al., “Treatment of experimental visceral leishmaniasis with amphotericin B in stable albumin microspheres,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 9, pp. 3246–3252, 2004. View at Publisher · View at Google Scholar · View at Scopus
  37. O. Cortadellas, “Initial and long-term efficacy of a lipid emulsion of amphotericin B desoxycholate in the management of canine leishmaniasis,” Journal of Veterinary Internal Medicine, vol. 17, no. 6, pp. 808–812, 2003. View at Publisher · View at Google Scholar
  38. J. Moreno, J. Nieto, C. Chamizo et al., “The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy,” Veterinary Immunology and Immunopathology, vol. 71, no. 3-4, pp. 181–195, 1999. View at Publisher · View at Google Scholar · View at Scopus
  39. R. Espada, S. Valdespina, G. Molero, M. A. Dea, M. P. Ballesteros, and J. J. Torrado, “Efficacy of alternative dosing regimens of poly-aggregated amphotericin B,” International Journal of Antimicrobial Agents, vol. 32, no. 1, pp. 55–61, 2008. View at Publisher · View at Google Scholar · View at Scopus
  40. K. Bartlett, E. Yau, S. C. Hartsel et al., “Effect of heat-treated amphotericin B on renal and fungal cytotoxicity,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 1, pp. 333–336, 2004. View at Publisher · View at Google Scholar · View at Scopus
  41. E. W. M. van Etten, W. van Vianen, P. Roovers, and P. Frederik, “Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice,” Antimicrobial Agents and Chemotherapy, vol. 44, no. 6, pp. 1598–1603, 2000. View at Publisher · View at Google Scholar · View at Scopus
  42. A. H. A. Mohamed-Ahmed, K. A. Les, K. Seifert, S. L. Croft, and S. Brocchini, “Noncovalent complexation of amphotericin-B with poly(α-glutamic acid),” Molecular Pharmaceutics, vol. 10, no. 3, pp. 940–950, 2013. View at Publisher · View at Google Scholar · View at Scopus
  43. R. Espada, S. Valdespina, M. A. Dea et al., “In vivo distribution and therapeutic efficacy of a novel amphotericin B poly-aggregated formulation,” Journal of Antimicrobial Chemotherapy, vol. 61, no. 5, pp. 1125–1131, 2008. View at Publisher · View at Google Scholar · View at Scopus
  44. F. Gaboriau, M. Chéron, C. Petit, and J. Bolard, “Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index,” Antimicrobial Agents and Chemotherapy, vol. 41, no. 11, pp. 2345–2351, 1997. View at Google Scholar · View at Scopus
  45. L. Hernández, A. Montoya, R. Checa et al., “Course of experimental infection of canine leishmaniosis: follow-up and utility of noninvasive diagnostic techniques,” Veterinary Parasitology, vol. 207, no. 1-2, pp. 149–155, 2015. View at Publisher · View at Google Scholar
  46. R. Espada, S. Valdespina, C. Alfonso, G. Rivas, M. P. Ballesteros, and J. J. Torrado, “Effect of aggregation state on the toxicity of different amphotericin B preparations,” International Journal of Pharmaceutics, vol. 361, no. 1-2, pp. 64–69, 2008. View at Publisher · View at Google Scholar · View at Scopus
  47. F. Mancianti and N. Meciani, “Specific serodiagnosis of canine leishmaniasis by indirect immunofluorescence, indirect hemagglutination, and counterimmunoelectrophoresis,” The American Journal of Veterinary Research, vol. 49, no. 8, pp. 1409–1411, 1988. View at Google Scholar · View at Scopus
  48. WHO, “Report of a meeting of the WHO Expert Committee on the control of leishmaniases,” WHO Technical Report Series, WHO, Geneva, Switzerland, 2010. View at Google Scholar
  49. J. P. Dedet, F. Pratlong, R. Pradinaud, and B. Moreau, “Delayed culture of Leishmania in skin biopsies,” Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 93, no. 6, pp. 673–674, 1999. View at Publisher · View at Google Scholar · View at Scopus
  50. O. Francino, L. Altet, E. Sánchez-Robert et al., “Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis,” Veterinary Parasitology, vol. 137, no. 3-4, pp. 214–221, 2006. View at Publisher · View at Google Scholar · View at Scopus
  51. F. Dantas-Torres, M. S. Latrofa, and D. Otranto, “Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus sanguineus,” Parasites and Vectors, vol. 4, no. 1, article 56, 2011. View at Publisher · View at Google Scholar · View at Scopus
  52. M. Hommel, C. L. Jaffe, B. Travi, and G. Milon, “Experimental models for leishmaniasis and for testing anti-leishmanial vaccines,” Annals of Tropical Medicine and Parasitology, vol. 89, supplement 1, pp. 55–73, 1995. View at Google Scholar · View at Scopus
  53. J. Fernández-Cotrina, V. Iniesta, S. Belinchón-Lorenzo et al., “Experimental model for reproduction of canine visceral leishmaniosis by Leishmania infantum,” Veterinary Parasitology, vol. 192, no. 1–3, pp. 118–128, 2013. View at Publisher · View at Google Scholar · View at Scopus
  54. J. Poot, M. E. Rogers, P. A. Bates, and A. Vermeulen, “Detailed analysis of an experimental challenge model for Leishmania infantum (JPC strain) in dogs,” Veterinary Parasitology, vol. 130, no. 1-2, pp. 41–53, 2005. View at Publisher · View at Google Scholar · View at Scopus
  55. B. L. Travi, E. Y. Osorio, O. A. Saldarriaga et al., “Clinical, parasitologic, and immunologic evolution in dogs experimentally infected with sand fly-derived Leishmania chagasi promastigotes,” The American Journal of Tropical Medicine and Hygiene, vol. 81, no. 6, pp. 994–1003, 2009. View at Publisher · View at Google Scholar · View at Scopus
  56. A. Rodríguez-Cortés, A. Ojeda, L. López-Fuertes et al., “A long term experimental study of canine visceral leishmaniasis,” International Journal for Parasitology, vol. 37, no. 6, pp. 683–693, 2007. View at Publisher · View at Google Scholar · View at Scopus
  57. L. Solano-Gallego, A. Koutinas, G. Miró et al., “Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis,” Veterinary Parasitology, vol. 165, no. 1-2, pp. 1–18, 2009. View at Publisher · View at Google Scholar · View at Scopus
  58. J. A. Sánchez-Brunete, M. A. Dea, S. Rama et al., “Amphotericin B molecular organization as an essential factor to improve activity/toxicity ratio in the treatment of visceral leishmaniasis,” Journal of Drug Targeting, vol. 12, no. 7, pp. 453–460, 2004. View at Publisher · View at Google Scholar · View at Scopus
  59. D. R. Serrano, L. Hernández, L. Fleire et al., “Hemolytic and pharmacokinetic studies of liposomal and particulate amphotericin B formulations,” International Journal of Pharmaceutics, vol. 447, no. 1-2, pp. 38–46, 2013. View at Publisher · View at Google Scholar · View at Scopus
  60. I. Bekersky, G. W. Boswell, R. Hiles, R. M. Fielding, D. Buell, and T. J. Walsh, “Safety and toxicokinetics of intravenous liposomal amphotericin B (AmBisome) in beagle dogs,” Pharmaceutical Research, vol. 16, no. 11, pp. 1694–1701, 1999. View at Publisher · View at Google Scholar · View at Scopus
  61. M. Pierantozzi, X. Roura, S. Paltrinieri, M. Poggi, and A. Zatelli, “Variation of proteinuria in dogs with leishmaniasis treated with meglumine antimoniate and allopurinol: a retrospective study,” Journal of the American Animal Hospital Association, vol. 49, no. 4, pp. 231–236, 2013. View at Publisher · View at Google Scholar · View at Scopus
  62. S. Martínez-Subiela, L. J. Bernal, and J. J. Cerón, “Serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term treatment,” The American Journal of Veterinary Research, vol. 64, no. 8, pp. 1021–1026, 2003. View at Publisher · View at Google Scholar · View at Scopus
  63. L. V. Athanasiou, M. N. Saridomichelakis, V. I. Kontos, G. Spanakos, and T. S. Rallis, “Treatment of canine leishmaniosis with aminosidine at an optimized dosage regimen: a pilot open clinical trial,” Veterinary Parasitology, vol. 192, no. 1–3, pp. 91–97, 2013. View at Publisher · View at Google Scholar · View at Scopus
  64. V. Woerly, L. Maynard, A. Sanquer, and H.-M. Eun, “Clinical efficacy and tolerance of miltefosine in the treatment of canine leishmaniosis,” Parasitology Research, vol. 105, no. 2, pp. 463–469, 2009. View at Publisher · View at Google Scholar · View at Scopus
  65. J. Carcelén, V. Iniesta, J. Fernández-Cotrina et al., “The chimerical multi-component Q protein from Leishmania in the absence of adjuvant protects dogs against an experimental Leishmania infantum infection,” Vaccine, vol. 27, no. 43, pp. 5964–5973, 2009. View at Publisher · View at Google Scholar · View at Scopus
  66. A. C. Rosypal, G. C. Troy, R. B. Duncan, A. M. Zajac, and D. S. Lindsay, “Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum,” Journal of Veterinary Internal Medicine, vol. 19, no. 6, pp. 802–809, 2005. View at Google Scholar · View at Scopus
  67. P. Abranches, G. Santos-Gomes, N. Rachamim, L. Campino, L. F. Schnur, and C. L. Jaffe, “An experimental model for canine visceral leishmaniasis,” Parasite Immunology, vol. 13, no. 5, pp. 537–550, 1991. View at Publisher · View at Google Scholar · View at Scopus
  68. E. Pinelli, R. Killick-Kendrick, J. Wagenaar, W. Bernadina, G. Del Real, and J. Ruitenberg, “Cellular and humoral immune responses in dogs experimentally and naturally infected with Leishmania infantum,” Infection and Immunity, vol. 62, no. 1, pp. 229–235, 1994. View at Google Scholar · View at Scopus
  69. A. B. Reis, O. A. Martins-Filho, A. Teixeira-Carvalho et al., “Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis,” Research in Veterinary Science, vol. 81, no. 1, pp. 68–75, 2006. View at Publisher · View at Google Scholar · View at Scopus
  70. S. Rougier, L. Hasseine, P. Delaunay, G. Michel, and P. Marty, “One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis,” Veterinary Parasitology, vol. 186, no. 3-4, pp. 245–253, 2012. View at Publisher · View at Google Scholar · View at Scopus
  71. A. F. Koutinas, M. N. Saridomichelakis, M. E. Mylonakis et al., “A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis,” Veterinary Parasitology, vol. 98, no. 4, pp. 247–261, 2001. View at Publisher · View at Google Scholar · View at Scopus
  72. I. Amusategui, A. Sainz, and M. A. Tesouro, “Effects of antimonial therapy for canine leishmaniasis on antibody titer,” Annals of the New York Academy of Sciences, vol. 849, pp. 444–446, 1998. View at Publisher · View at Google Scholar · View at Scopus
  73. M. Torres, M. Bardagí, X. Roura, G. Zanna, I. Ravera, and L. Ferrer, “Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and treated with meglumine antimoniate and allopurinol,” Veterinary Journal, vol. 188, no. 3, pp. 346–351, 2011. View at Publisher · View at Google Scholar · View at Scopus
  74. L. Ferrer, M. J. Aisa, X. Roura, and M. Portús, “Serological diagnosis and treatment of canine leishmaniasis,” Veterinary Record, vol. 136, no. 20, pp. 514–516, 1995. View at Publisher · View at Google Scholar · View at Scopus
  75. L. Solano-Gallego, P. Morell, M. Arboix, J. Alberola, and L. Ferrer, “Prevalence of Leishmania infantum infection in dogs living in an area of canine Leishmaniasis endemicity using PCR on several tissues and serology,” Journal of Clinical Microbiology, vol. 39, no. 2, pp. 560–563, 2001. View at Publisher · View at Google Scholar · View at Scopus
  76. V. Martínez, J. Quilez, A. Sanchez, X. Roura, O. Francino, and L. Altet, “Canine leishmaniasis: the key points for qPCR result interpretation,” Parasites and Vectors, vol. 4, article 57, 2011. View at Publisher · View at Google Scholar · View at Scopus
  77. J. Alvar, C. Cañavate, R. Molina, J. Moreno, and J. Nieto, “Canine leishmaniasis,” Advances in Parasitology, vol. 57, pp. 1–88, 2004. View at Publisher · View at Google Scholar · View at Scopus
  78. L. Ferrer, “Clinical aspects of canine leishmaniosis: an update,” in Proceedings of the International Canine Leishmaniasis Forum, pp. 6–10, Hoechst Roussel Vet, Barcelona, Spain, 1999.
  79. C. Maia and L. Campino, “Methods for diagnosis of canine leishmaniasis and immune response to infection,” Veterinary Parasitology, vol. 158, no. 4, pp. 274–287, 2008. View at Publisher · View at Google Scholar · View at Scopus
  80. P. F. Quaresma, S. M. F. Murta, E. de Castro Ferreira, A. C. V. M. da Rocha-Lima, A. A. P. Xavier, and C. M. F. Gontijo, “Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR,” Acta Tropica, vol. 111, no. 3, pp. 289–294, 2009. View at Publisher · View at Google Scholar · View at Scopus
  81. L. Manna, S. Reale, E. Viola et al., “Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs,” Veterinary Parasitology, vol. 142, no. 3-4, pp. 271–280, 2006. View at Publisher · View at Google Scholar · View at Scopus
  82. L. Manna, S. Reale, E. Picillo, F. Vitale, and A. E. Gravino, “Urine sampling for real-time polymerase chain reaction-based diagnosis of canine leishmaniasis,” Journal of Veterinary Diagnostic Investigation, vol. 20, no. 1, pp. 64–67, 2008. View at Publisher · View at Google Scholar · View at Scopus
  83. L. Solano-Gallego, A. Rodriguez-Cortes, M. Trotta et al., “Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis,” Veterinary Parasitology, vol. 147, no. 3-4, pp. 315–319, 2007. View at Publisher · View at Google Scholar · View at Scopus
  84. A. Franceschi, V. Merildi, G. Guidi, and F. Mancianti, “Occurrence of leishmania DNA in urines of dogs naturally infected with leishmaniasis,” Veterinary Research Communications, vol. 31, no. 3, pp. 335–341, 2007. View at Publisher · View at Google Scholar · View at Scopus