Genetic diversity of Cryptosporidium spp. in captive reptiles

Abstract

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.

FIG. 1.

Sequence diversity in the SSU rRNA gene among Cryptosporidium in reptiles. Dots denote sequence identity to isolate 18, dashes represent nucleotide deletions. Isolate designations: 18, C. serpentis genotype A; 63, C. serpentis genotype B; 1665, a C. serpentis-like Cryptosporidium in lizard 1665; 1779, C. muris; 750, Cryptosporidium tortoise genotype; 1432, C. parvum bovine genotype; 1444, C. parvum mouse genotype; 2162, another new Cryptosporidium genotype in snake 2162; 815, C. saurophilum; 938, a new Cryptosporidium genotype in snake 938.

FIG. 2.

Genetic relationship between Cryptosporidium spp. in reptiles inferred by a neighbor-joining analysis of the partial SSU rRNA gene sequences by using the Kimura two-parameter model and the Treecon program. Numbers on branches are percentage bootstrap values of 1,000 replicates. Only values above 50% are shown.

FIG. 3.

Morphology of C. saurophilum (A) and C. serpentis (B) as seen under a differential interference contrast microscope (magnification, ×1,000).

FIG. 4.

Simultaneous presence of multiple Cryptosporidium spp. in a group of six snakes and four lizards housed together as revealed by PCR-RFLP analyses of the SSU rRNA gene. The upper panel shows the results of SspI digestion; the lower panel shows the results of VspI digestion. Lanes 1 and 14, 100-bp molecular markers; lane 2, positive control for C. serpentis; lane 3, positive control for C. saurophilum; lane 4, sample from pine snake 936; lane 5, sample from pine snake 937; lane 6, sample from a New Guinea viper boa (938); lane 7, sample from a milk snake (939); lane 8, sample from a black rat snake (940); lane 9, sample from a green python (941); lane 10, sample from mountain chameleon 942; lane 11, sample from mountain chameleon 943; lane 12, sample from a bearded dragon (944); lane 13, sample from a gargoyle gecko (945). Filled and open arrows are the SspI and VspI bands, respectively, for the new Cryptosporidium genotype in snakes. Three Cryptosporidium spp. (C. serpentis, a new Cryptosporidium genotype, and a trace of C. saurophilum) are seen in all six snakes (lanes 4 to 9), and two parasites (C. serpentis and C. saurophilum) are seen in all 4 lizards (lanes 10 to 13).