Folia Parasitologica 63:035 (2016) | DOI: 10.14411/fp.2016.035

Cryptosporidium testudinis sp. n., Cryptosporidium ducismarci Traversa, 2010 and Cryptosporidium tortoise genotype III (Apicomplexa: Cryptosporidiidae) in tortoises

Jana Ježková^1,2, Michaela Horčičková^1,3, Lenka Hlásková¹, Bohumil Sak¹, Dana Květoňová¹, Jan Novák⁴, Lada Hofmannová⁵, John McEvoy⁶, Martin Kváč^1,3

¹ Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic;

² Faculty of Science, University of South Bohemia in České Budějovice, Czech Republic;

³ Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic;

⁴ Faculty of Fisheries and Protection of Waters, South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, Institute of Complex Systems, University of South Bohemia in České Budějovice, Czech Republic;

⁵ Department of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic;

⁶ Veterinary and Microbiological Sciences Department, North Dakota State University, Fargo, USA

Understanding of the diversity of species of Cryptosporidium Tyzzer, 1910 in tortoises remains incomplete due to the limited number of studies on these hosts. The aim of the present study was to characterise the genetic diversity and biology of cryptosporidia in tortoises of the family Testudinidae Batsch. Faecal samples were individually collected immediately after defecation and were screened for presence of cryptosporidia by microscopy using aniline-carbol-methyl violet staining, and by PCR amplification and sequence analysis targeting the small subunit rRNA (SSU), Cryptosporidium oocyst wall protein (COWP) and actin genes. Out of 387 faecal samples from 16 tortoise species belonging to 11 genera, 10 and 46 were positive for cryptosporidia by microscopy and PCR, respectively. All samples positive by microscopy were also PCR positive. Sequence analysis of amplified genes revealed the presence of the Cryptosporidium tortoise genotype I (n = 22), C. ducismarci Traversa, 2010 (n = 23) and tortoise genotype III (n = 1). Phylogenetic analyses of SSU, COWP and actin gene sequences revealed that Cryptosporidium tortoise genotype I and C. ducismarci are genetically distinct from previously described species of Cryptosporidium. Oocysts of Cryptosporidium tortoise genotype I, measuring 5.8-6.9 µm × 5.3-6.5 µm, are morphologically distinguishable from C. ducismarci, measuring 4.4-5.4 µm × 4.3-5.3 µm. Oocysts of Cryptosporidium tortoise genotype I and C. ducismarci obtained from naturally infected Russian tortoises (Testudo horsfieldii Gray) were infectious for the same tortoise but not for Reeve's turtles (Mauremys reevesii [Gray]), common garter snake (Thamnophis sirtalis [Linnaeus]), zebra finches (Taeniopygia guttata [Vieillot]) and SCID mice (Mus musculus Linnaeus). The prepatent period was 11 and 6 days post infection (DPI) for Cryptosporidium tortoise genotype I and C. ducismarci, respectively; the patent period was longer than 200 days for both cryptosporidia. Naturally or experimentally infected tortoises showed no clinical signs of disease. Our morphological, genetic, and biological data support the establishment of Cryptosporidium tortoise genotype I as a new species, Cryptosporidium testudinis sp. n., and confirm the validity of C. ducismarci as a separate species of the genus Cryptosporidium.

Keywords: morphology, transmission studies, taxonomy, new species, molecular phylogeny

Received: June 3, 2016; Accepted: September 9, 2016; Published online: October 14, 2016  Show citation