Molecular and Biochemical Parasitology 108 (2000) 187 – 197 Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parum G. Widmer a , D. Akiyoshi a , M.A. Buckholt a , X. Feng a , S.M. Rich a , K.M. Deary a , C.A. Bowman a , P. Xu b , Y. Wang b , X. Wang b , G.A. Buck b , S. Tzipori a, * a The Diision of Infectious Diseases, Tufts Uniersity School of Veterinary Medicine, North Grafton, MA 01536, USA b Microbiology and Immunology, Virginia Commonwealth Uniersity, Richmond, VA 23198, USA Received 15 November 1999; received in revised form 10 February 2000; accepted 14 February 2000 Abstract Human cryptosporidiosis is attributed to two major Cryptosporidium parum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium parum; Genotype; Gnotobiotic pig; Genome sequence survey; Restriction fragment length polymor- phism www.elsevier.com/locate/parasitology Abbreiations: GKO, interferon- knock-out; GSS, genome sequence survey; PI, post-inoculation; RFLP, restriction fragment length polymorphism. * Corresponding author. Tel.: +1-508-8397955; fax: +1-508-8397977. E-mail address: stzipori@infonet.tufts.edu (S. Tzipori) 0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0166-6851(00)00211-5