Veterinary Parasitology 204 (2014) 134–138 Contents lists available at ScienceDirect Veterinary Parasitology jou rn al hom epage : www.elsevier.com/locate/vetpar The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR Deuvânia C. da Silva a , Philipp Ricardo S.O. Paiva b , Alex Akira Nakamura a , Camila Guariz Homem a , Milena Sato de Souza a , Kathleen Fernandes Grego c , Marcelo Vasconcelos Meireles a,∗ a Faculdade de Medicina Veterinária, Univ Estadual Paulista, UNESP, Rua Clóvis Pestana, 793, Arac ¸ atuba, SP, Brazil b Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, USP, Avenida Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, SP, Brazil c Laboratório de Herpetologia, Instituto Butantan, Avenida Vital Brazil, 1500 São Paulo, SP, Brazil a r t i c l e i n f o Article history: Received 28 December 2013 Received in revised form 21 April 2014 Accepted 5 May 2014 Keywords: Epidemiology Cryptosporidium Reptiles Molecular diagnosis a b s t r a c t Infection by Cryptosporidium serpentis occurs in reptiles, particularly in snakes. This dis- ease is characterized by chronic infection with the presence of hypertrophic gastritis. The objectives of this study were to use real-time polymerase chain reaction (PCR) targeting the heat shock protein 70 (Hsp70) gene for the detection of C. serpentis in fecal samples from snakes and to determine the analytical and epidemiological specificity and sensitivity of this approach relative to the gold standard of nested PCR for the amplification of a frag- ment of the 18S subunit of the ribosomal RNA (18S rRNA) gene followed by the sequencing of amplified fragments (nPCR/S). Individual fecal samples were collected on a single occa- sion from 503 asymptomatic adult snakes housed in the serpentarium of the Butantan Institute in São Paulo, Brazil. The nested PCR revealed that 60 samples (11.98%) were pos- itive for Cryptosporidium sp. The sequencing of amplified fragments, which was possible for 38 samples, resulted in the identification of Cryptosporidium tyzzeri (7), Cryptosporidium muris (4), Cryptosporidium varanii (12) and C. serpentis (15) in fecal samples from several snake species. The real-time PCR approach indicated that 17 samples (3.37%) were positive for C. serpentis, whereas the nPCR/S indicated that 15 samples (2.98%) were positive for C. serpentis. The epidemiological sensitivity and specificity of real-time PCR were 93.8% and 99.5%, respectively. Thus, we conclude that real-time PCR targeting the Hsp70 gene is a sensitive and specific method for the detection of C. serpentis in snake fecal samples. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Cryptosporidium is a coccidian protozoan of the phylum Apicomplexa that infect amphibians, birds, fishes, mam- mals and reptiles. Three valid species of Cryptosporidium ∗ Corresponding author. Tel.: +55 18 3636 1425/+55 18 981111836; fax: +55 18 3636 1403. E-mail addresses: marcelo@fmva.unesp.br, meirelesmv@uol.com.br (M.V. Meireles). infect reptiles: Cryptosporidium ducismarci, associated with intestinal pathology in tortoises, Cryptosporidium varanii (a replacement for the junior synonym Cryptosporidium saurophilum), which is most frequently found in the intesti- nal epithelium of lizards, and Cryptosporidium serpentis, which is found in the gastric epithelium of snakes (Koudela and Modr ´ y, 1998; Pavlasek and Ryan, 2008; Traversa, 2010; ˇ Slapeta, 2013). C. serpentis infection occurs in snakes and mani- fests as chronic hypertrophic gastritis with inappetence, http://dx.doi.org/10.1016/j.vetpar.2014.05.012 0304-4017/© 2014 Elsevier B.V. All rights reserved.