Rapid displacement of Cryptosporidium parvum type 1 by type 2 in mixed infections in piglets

Abstract

Genotypes 1 and 2 of Cryptosporidium parvum are the primary types associated with infections in humans, with type 1 being by far the predominant genotype. The frequency of mixed infection with both genotypes in humans is relatively rare, while type 1, which experimentally infects other mammals, has been found to naturally infect almost exclusively humans. One possible explanation for the absence of type 1 in other mammals and the low frequency of mixed infections in humans is the inability of type 1 to compete with type 2 in nature when both occur simultaneously. To investigate this, we challenged gnotobiotic piglets with equal number of oocysts of type 1 and type 2, given either simultaneously or with type 2 given 24 or 48 h after type 1. The genotype of the oocysts excreted in feces and the relative distribution of each of the genotypes throughout the gut at necropsy were determined. Regardless of the time interval between challenges with the two genotypes, type 2 invariably displaced type 1. The rate of displacement was rapid when both genotypes were given simultaneously, after which no traces of type 1 were detected in the feces or in gut sections by PCR. Infection with type 1 24 or 48 h before challenge with type 2, while permitting type 1 to become established, was still rapidly eliminated within 3 days after challenge with type 2. These observations have major implications regarding the relative perpetuation and survival of these two genotypes in mammals.

FIG. 1.

Genotype analysis of fecal samples from the gnotobiotic piglets. COWP PCR was performed, and the PCR products were digested with RsaI for 90 min at 37°C. The RsaI digests were electrophoresed on a 15% Tris-borate-EDTA acrylamide gel and were visualized by ethidium bromide staining. The samples are as follows: 100-bp ladder (lanes 1 and 31 [Promega Corp.]); type 1 control (lane 2); type 2 control (lane 3); piglet 1 (type 1), day 4 before noon (lane 4), day 4 after noon (lane 5), and day 5 before noon (lane 6); piglet 2 (type 2), day 3 (lane 7), day 4 before noon (lane 8), day 4 after noon (lane 9), and day 5 before noon (lane 10); piglet 3 (mixed), day 3 before noon (lane 11), day 4 before noon (lane 12), day 4 after noon (lane 13), and day 5 before noon (lane 14); piglet 4 (mixed, type 2 challenge on day 1, euthanized day 4), day 3 before noon (lane 15), day 3 after noon (lane 16), and day 4 before noon (lane 17); piglet 5 (mixed, type 2 challenge on day 1, euthanized day 6), day 3 before noon (lane 18), day 3 after noon (lane 19), day 4 before noon (lane 20), day 4 after noon (lane 21), day 5 before noon (lane 22), day 5 after noon (lane 23), and day 6 before noon (lane 24); piglet 6 (mixed, type 2 challenge on day 2, euthanized day 4), day 3 before noon (lane 25), and day 4 before noon (lane 26); piglet 7 (mixed, type 2 challenge day 2, euthanized day 6), day 3 before noon (lane 27), day 4 after noon (lane 28), day 5 before noon (lane 29), and day 6 before noon (lane 30). Digestion with RsaI yields fragments of 285, 125, 106, and 34 bp for type 1 and 410, 106, and 34 bp for type 2. Samples showing fragments of 410, 285, 125, 106, and 34 bp are indicative of a mixed infection with both type 1 and type 2 present.

FIG. 2.

Genotype analysis of the intestinal sections from infected gnotobiotic piglets by COWP PCR-RFLP. Sections were obtained at necropsy and extend from the stomach (left) to the distal spiral colon (right). (A) Piglet 1 (type 1; lanes 4 to 18), piglet 2 (type 2; lanes 20 to 32), piglet 3 (mixed; lanes 34 to 49), and 100-bp ladder (lanes 1, 19, 33, and 50). (B) Piglet 4 (mixed, type 2 day 1, euthanized day 4; lanes 4 to 18), piglet 5 (mixed, type 2 day 1, euthanized day 6; lanes 20 to 34), and 100-bp ladder (lanes 1, 19, and 35). (C) Piglet 6 (mixed, type 2 day 2, euthanized day 4; lanes 4 to 14), piglet 7 (mixed, type 2 day 2, euthanized day 6; lanes 16 to 30), and 100-bp ladder (lanes 1, 15, and 31). Type 1 and type 2 controls are in lanes 2 and 3 of each panel, respectively.

FIG. 3.

Anatomical site, histology scores, and genotyping results of intestinal samples obtained from piglets at necropsy: piglet 1 (type 1 only); piglet 2 (type 2 only), piglet 3 (type 1 and type 2 challenge on day 0), piglets 4 and 5 (mixed infection with type 2 challenge on day 1), and piglets 6 and 7 (mixed infections with type 2 challenge on day 2). Piglets 1 through 3 were euthanized on day 5, piglets 4 and 6 were euthanized on day 4, and piglets 5 and 7 were euthanized on day 6. Sections are numbered consecutively from stomach to distal spiral colon (left to right). Histology scoring is as follows: 0, no parasites detected on intestinal villi; 1, few parasites detected; 2, 25% of villus surface covered by parasites; 3, 50% of villus surface covered by parasites; 4, 75% of villus surface covered by parasites; and 5, 100% of villus surface covered by parasites. S, stomach; SI, small intestine; LI, large intestine; ▨ band, intestinal sample positive for type 1 by COWP PCR-RFLP; ▪ band, intestinal sample positive for type 2 by COWP PCR-RFLP; and na, genotyping results not available.

FIG. 4.

Cultured MDBK cells were infected with 50,000 oocysts/ml (type 2, types 1 and 2, or type 1). Cells were harvested as follows: type 2, 6 h (lane 3), 11 h (lane 4), 24 h (lane 5), and 48 h postinfection (lane 6) and heat inactivated (lane 7); types 1 and 2, 6 h (lane 8), 11 h (lane 9), 24 h (lane 10), 48 h (lane 11), and heat inactivated (lane 12); type 1, 6 h (lane 13), 11 h (lane 14), 24 h (lane 15), 48 h (lane 16), and heat inactivated (lane 17). COWP PCR-RFLP analysis was as previously described. Type 2 control (lane 2) and 100-bp ladder (Promega Corp.) (lane 1) are also shown. When both genotypes appear in the mixed-infection wells, there is a suggestion that type 2 is more predominant than when either genotype is alone, based on intensity of the PCR product. The heat-inactivated oocyst samples are representative of the oocyst populations that were added to the culture, in the absence of replication.