Cryptosporidium proliferans n. sp. (Apicomplexa: Cryptosporidiidae): Molecular and Biological Evidence of Cryptic Species within Gastric Cryptosporidium of Mammals

Abstract

The morphological, biological, and molecular characteristics of Cryptosporidium muris strain TS03 are described, and the species name Cryptosporidium proliferans n. sp. is proposed. Cryptosporidium proliferans obtained from a naturally infected East African mole rat (Tachyoryctes splendens) in Kenya was propagated under laboratory conditions in rodents (SCID mice and southern multimammate mice, Mastomys coucha) and used in experiments to examine oocyst morphology and transmission. DNA from the propagated C. proliferans isolate, and C. proliferans DNA isolated from the feces of an African buffalo (Syncerus caffer) in Central African Republic, a donkey (Equus africanus) in Algeria, and a domestic horse (Equus caballus) in the Czech Republic were used for phylogenetic analyses. Oocysts of C. proliferans are morphologically distinguishable from C. parvum and C. muris HZ206, measuring 6.8-8.8 (mean = 7.7 μm) × 4.8-6.2 μm (mean = 5.3) with a length to width ratio of 1.48 (n = 100). Experimental studies using an isolate originated from T. splendens have shown that the course of C. proliferans infection in rodent hosts differs from that of C. muris and C. andersoni. The prepatent period of 18-21 days post infection (DPI) for C. proliferans in southern multimammate mice (Mastomys coucha) was similar to that of C. andersoni and longer than the 6-8 DPI prepatent period for C. muris RN66 and HZ206 in the same host. Histopatologicaly, stomach glands of southern multimammate mice infected with C. proliferans were markedly dilated and filled with necrotic material, mucus, and numerous Cryptosporidium developmental stages. Epithelial cells of infected glands were atrophic, exhibited cuboidal or squamous metaplasia, and significantly proliferated into the lumen of the stomach, forming papillary structures. The epithelial height and stomach weight were six-fold greater than in non-infected controls. Phylogenetic analyses based on small subunit rRNA, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70, actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences revealed that C. proliferans is genetically distinct from C. muris and other previously described Cryptosporidium species.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Cryptosporidium proliferans, Cryptosporidium muris HZ206, and Cryptosporidium parvum oocysts in (A) differential interference contrast microscopy and stained by (B) aniline–carbol–methyl violet (C) Auramine Phenol and (D) anti-Cryptosporidium FITC-conjugated antibody.

Bar = 10 μm.

Fig 2. Phylogenetic relationships between Cryptosporidium proliferans (highlighted) and other Cryptosporidium spp. as inferred by a neighbor-joining analysis (NJ)/maximum parsimony(MP)/maximum likelihood (ML) of (A) the SSU (706 base positions in the final dataset; ML = log -2886.67) and (B) TRAP-C1 (531 base positions in the final dataset, ML = log -1929.25).

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Scale bar included in each tree.

Fig 3. Phylogenetic relationships between Cryptosporidium proliferans (highlighted) and other Cryptosporidium spp. as inferred by a neighbor-joining analysis (NJ)/maximum parsimony(MP)/maximum likelihood (ML) of (A) HSP70 (211 base positions in the final dataset, ML = log -1745.42) and (B) COWP (369 base positions in the final dataset, ML = log -532.78).

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Scale bar included in each tree.

Fig 4. Phylogenetic relationships between Cryptosporidium proliferans (highlighted) and other Cryptosporidium spp. as inferred by a neighbor-joining analysis (NJ)/maximum parsimony (MP)/maximum likelihood (ML) of (A) actin (728 base positions in the final dataset, ML = log = -5522.35) and (B) MS1 (436 base positions in the final dataset, ML = log -886.75). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates).

Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Interrupted branches have been shortened five-fold. Scale bar included in each tree.

Fig 5. Phylogenetic relationships between Cryptosporidium proliferans (highlighted) and other Cryptosporidium spp. as inferred by a neighbor-joining analysis (NJ)/maximum parsimony (MP)/maximum likelihood (ML) of (A) MS2 (442 base positions in the final dataset, ML = log -792.33), (B) MS3 (485 base positions in the final dataset, ML = log = -832.45), and (C) MS16 (580 base positions in the final dataset, ML = log = -956.78).

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Scale bar included in each tree.

Fig 6. Phylogenetic relationships between Cryptosporidium proliferans and selected Cryptosporidium spp. as inferred by a neighbor-joining analysis (NJ)/maximum parsimony(MP)/maximum likelihood (ML) analysis of a concatenated sequence constructed from partial DNA sequences of SSU, actin, COWP, HSP70, and TRAP-C1 genes (1991 base positions in the final dataset, ML = log = -4368.98).

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Scale bar included in tree.

Fig 7. Course of infection of Cryptosporidium proliferans and Cryptosporidium muris HZ206 in Mastomys coucha based on coprological examination of feces.

Fig 8. Height of mucosa of Mastomys coucha stomach of A) control group, B) Cryptosporidium muris HZ206 infection, and C) Cryptosporidium proliferans infection at 28 day post infection; D) stomach mucosa proliferation in Mastomys coucha with Cryptosporidium proliferans at 140 DPI.

Haematoxylin and eosin. Bar = 150 μm.

Fig 9. Change of stomach weight of Mastomys coucha during experimental infection with Cryptosporidium proliferans and Cryptosporidium muris HZ206 compared to the control group.