Characterization of Cryptosporidium meleagridis of human origin passaged through different host species

Abstract

Cryptosporidium meleagridis, a protozoon first observed in turkeys, has been linked by several investigators to cryptosporidiosis in humans. C. meleagridis is the only known Cryptosporidium species that infects both avian and mammalian species. We describe the successful propagation of C. meleagridis (isolate TU1867), originally purified from a patient with diarrhea, in laboratory animals including chickens, mice, piglets, and calves. TU1867 was readily transmitted from one animal host to another, maintaining genetic homogeneity and stability. The rate of infectivity and virulence of TU1867 for the mammalian species were similar to those of Cryptosporidium parvum. Laboratory propagation of genetically and phenotypically stable and well-characterized reference isolates, representing various Cryptosporidium species, particularly those infectious to humans, will improve considerably the spectrum and quality of laboratory and field investigations on this medically important protozoa.

FIG. 1.

Electron micrographs of sections from a mouse (A to F and H) and a chicken (G) experimentally infected with C. meleagridis. (A) Early development of a trophozoite as it is being engulfed by the parasitophorous membrane. (B) Fully developed trophozoite. (C) First-generation schizogony with 8 merozoites ready to be released into the intestinal lumen. (D) Second-generation schizogony with 4 merozoites which will develop into male or female sexual stages. (E) Dividing microgamy, showing 6 of 16 developing microgametes, the male sexual stage. (F) Fertilized macrogamete or zygote. (G) Fertilized macrogamete or zygote from an infected chicken, showing similar morphology and developmental stage to that of a zygote grown in a mouse (F). (H) Fully developed oocyst still within its parasitophorous vesicle. Bar, 1 μm.

FIG. 2.

COWP (A) and SSU rRNA (B) PCR-RFLP analyses of oocysts from C. meleagridis TU1867 passaged through different host species. For COWP and SSU rRNA RFLP analysis, PCR products were digested with RsaI and AseI, respectively. Samples are from the following animal passages: human (lane 2), first mouse (lane 3), last mouse (lane 4), first pig (lane 5), last pig (lane 6), first chicken (lane 7), last chicken (lane 8), first turkey (lane 9), last turkey (lane 10), and calf (lane 11). C. parvum genotype 1 (TU502) and genotype 2 (GCH1) digested PCR products are shown in lanes 12 and 13, respectively. A negative PCR control (lane 14), uncut PCR product (lane 15), and 100-bp DNA ladder (lanes 1 and 16; Promega Corp., Madison, Wis.) are also included. The mock DNA extraction sample was negative for both PCRs (data not shown). The expected sizes of the 553-bp RsaI-digested C. meleagridis COWP fragment are 372, 147, and 34 bp, and the sizes of the 833-bp AseI-digested SSU rRNA fragment are 456, 171, 104, and 102 bp. The expected sizes of this COWP fragment are 284, 129, 106, and 34 bp for C. parvum genotype 1 and 413, 106, and 34 bp for C. parvum genotype 2. The expected sizes of the SSU rRNA fragment are 561, 104, 102, and 70 bp for C. parvum genotype 1 (837 bp, AF481962) and 628, 104, and 102 bp for genotype 2 (834 bp, AF164102).