Please cite this article in press as: Moreno, I., et al., Detection of anti-Leishmania infantum antibodies in sylvatic lago- morphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.10.010 ARTICLE IN PRESS G Model VETPAR-7002; No. of Pages 4 Veterinary Parasitology xxx (2013) xxx–xxx Contents lists available at ScienceDirect Veterinary Parasitology jo u r nal homep age: www.elsevier.com/locate/vetpar Short Communication Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test Inmaculada Moreno a , Julio Álvarez b , Nerea García c , Santiago de la Fuente d , Irene Martínez c , Eloy Marino d , Alfredo Tora ˜ no a,∗ , Joaquin Goyache e , Felipe Vilas d , Lucas Domínguez c,e , Mercedes Domínguez a a Área de Inmunología, Unidad de Inmunología Microbiana e Inmunogenética, Instituto de Salud Carlos III, Mahadahonda, 28220 Madrid, Spain b Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), 28034 Madrid, Spain c Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, 28040 Madrid, Spain d Dirección General de Ordenación e Inspección, Consejería de Sanidad de la Comunidad de Madrid, 28001 Madrid, Spain e Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense Madrid, 28040 Madrid, Spain a r t i c l e i n f o Article history: Received 3 May 2013 Received in revised form 7 October 2013 Accepted 15 October 2013 Keywords: Leishmaniosis Immunofluorescence Incidental reservoirs Hare Anti-Leishmania antibodies Promastigote epitope expression a b s t r a c t An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infec- tion reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreac- tivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections. © 2013 Elsevier B.V. All rights reserved. The leishmanioses are a group of vector-borne diseases caused by parasites of the genus Leishmania (Ba˜ nuls et al., 2007). Infection is transmitted through the bite of infected phlebotomine sandflies which inoculate promastigotes in host skin while probing for a blood meal (Bates, 2007). Most leishmanioses are zoonotic, and in rural areas Leismania maintains persistent foci by preferentially infecting cer- tain mammalian hosts as primary, or infection reservoirs (Ashford, 1996). Depending on environmental condi- tions, Leishmania can perpetuate their endemic cycle by ∗ Corresponding author. Tel.: +34 91 822 3610; fax: +34 91 822 3423. E-mail address: atorano@isciii.es (A. Tora ˜ no). infecting other hosts, termed incidental or secondary reser- voirs, which act as “liaisons” with the final host in the transmission chain (Ashford, 2003). In Mediterranean countries, the Americas, the Mid- dle East and China, Leishmania infantum is the etiological agent of visceral and cutaneous leishmaniosis (Boelaert et al., 2000). In most areas where L. infantum is endemic, dogs are the main peridomestic reservoir (Baneth et al., 2008), although mammals other than dogs can be natu- rally infected and can have a role as secondary reservoirs (Gramiccia, 2011); in the Iberian peninsula, these include the black rat (Morillas Márquez et al., 1985), horse (Solano- Gallego et al., 2003), cat (Martín-Sánchez et al., 2007; Ayllón et al., 2008, 2012), red fox (Criado-Fornelio et al., 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.10.010