Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris)

Abstract

Background: Cryptosporidium spp. are globally distributed parasites that infect epithelial cells in the microvillus border of the gastrointestinal tract of all classes of vertebrates. Cryptosporidium chipmunk genotype I is a common parasite in North American tree squirrels. It was introduced into Europe with eastern gray squirrels and poses an infection risk to native European squirrel species, for which infection is fatal. In this study, the biology and genetic variability of different isolates of chipmunk genotype I were investigated.

Methods: The genetic diversity of Cryptosporidium chipmunk genotype I was analyzed by PCR/sequencing of the SSU rRNA, actin, HSP70, COWP, TRAP-C1 and gp60 genes. The biology of chipmunk genotype I, including oocyst size, localization of the life cycle stages and pathology, was examined by light and electron microscopy and histology. Infectivity to Eurasian red squirrels and eastern gray squirrels was verified experimentally.

Results: Phylogenic analyses at studied genes revealed that chipmunk genotype I is genetically distinct from other Cryptosporidium spp. No detectable infection occurred in chickens and guinea pigs experimentally inoculated with chipmunk genotype I, while in laboratory mice, ferrets, gerbils, Eurasian red squirrels and eastern gray squirrels, oocyst shedding began between 4 and 11 days post infection. While infection in mice, gerbils, ferrets and eastern gray squirrels was asymptomatic or had mild clinical signs, Eurasian red squirrels developed severe cryptosporidiosis that resulted in host death. The rapid onset of clinical signs characterized by severe diarrhea, apathy, loss of appetite and subsequent death of the individual may explain the sporadic occurrence of this Cryptosporidium in field studies and its concurrent spread in the population of native European squirrels. Oocysts obtained from a naturally infected human, the original inoculum, were 5.64 × 5.37 μm and did not differ in size from oocysts obtained from experimentally infected hosts. Cryptosporidium chipmunk genotype I infection was localized exclusively in the cecum and anterior part of the colon.

Conclusions: Based on these differences in genetics, host specificity and pathogenicity, we propose the name Cryptosporidium mortiferum n. sp. for this parasite previously known as Cryptosporidium chipmunk genotype I.

Keywords: Biology; Course of infection; Cryptosporidiosis; Genetic diversity; Mortality; Oocyst size; Phylogeny.

Fig. 1

Evolutionary relationships of Cryptosporidium spp. at the small subunit rRNA locus (SSU) inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to supported node. The GenBank accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 2

Evolutionary relationships of Cryptosporidium spp. at the 60 kDa glycoprotein locus (gp60) inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to the supported node. The GenBank accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 3

Evolutionary relationships of Cryptosporidium spp. at the actin locus inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to supported node. The GenBank accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 4

Evolutionary relationships of Cryptosporidium spp. at the Cryptosporidium oocyst wall protein (COWP) locus inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. The evolutionary distances were computed using the General Time Reversible model with a gamma distribution. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to supported node. The GenBank Accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 5

Evolutionary relationships of Cryptosporidium spp. at the 70-kDa heat shock protein (HSP70) inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to supported node. The GenBank Accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 6

Evolutionary relationships of Cryptosporidium spp. at the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) locus inferred using the maximum likelihood (ML)/neighbor-joining (NJ) method. Percentage supports (> 50%) from 1000 pseudoreplicates from ML and NJ analysis, respectively, are indicated next to supported node. The GenBank accession number is in parentheses. Sequences obtained in this study are identified by isolate number (e.g. 14762) and highlighted

Fig. 7

Course of infection of Cryptosporidium chipmunk genotype I. in different strains of experimentally inoculated laboratory mice (Mus musculus), gerbils (Meriones unquiculatus), ferrets (Mustela putorius furo), Eurasian red squirrels (Sciurus vulgaris) and eastern gray squirrels (Sciurus carolinensis). a Infection intensity expressed as number of oocysts per gram of feces (OPG) and b detection of oocysts based on molecular and microscopic examination of fecal samples. Black circles indicate the presence of oocysts and specific DNA of Cryptosporidium chipmunk genotype I; white circles indicate detection of specific-DNA only without oocyst detection. Crosses indicate that an animal was killed because of poor health

Fig. 8

Oocysts of Cryptosporidium chipmunk genotype I a in differential interference contrast microscopy, b stained by aniline-carbol-methyl violet staining, c stained by Ziehl-Nielsen staining, d stained by auramine-phenol staining and e labeled with anti-Cryptosporidium FITC-conjugated antibody. Bars = 5 μm

Fig. 9

Scanning electron microphotograph showing developmental stages of Cryptosporidium chipmunk genotype I on cecal mucosal epithelium in experimentally infected Eurasian red squirrel (Sciurus vulgaris) killed 16 days post infection (DPI) (a–c) and SCID mouse (Mus musculus) killed 30 DPI (d–f). a and d Surface of cecum covered with developmental stages, b released zoite (z); c merozoites (me) budding from residual body (rb); e zoites invading host tissue (z) with formation of merozoites covered with parasitophorous sac (me) and surrounded by elongated microvilli (mi), f mature meront with fully developed merozoites (me) with recognizable apical part (ap). Scale bars included in each figure

Fig. 10

Histology sections of the cecum of Eurasian red squirrel (Sciurus vulgaris) (a, b) and SCID mouse (Mus musculus) (c, d) experimentally infected with Cryptosporidium chipmunk genotype I and killed 16 and 30 days post infection, respectively. Attached developmental stages indicated by arrowhead. Periodic acid-Schiff (PAS) staining. Scale bar included in each figure

Fig. 11

Developmental stages of Cryptosporidium chipmunk genotype I in mucosal smears obtained from the cecum of SCID mouse (Mus musculus) experimentally infected with 100,000 oocysts and killed 30 days post infection. a Oocyst; b sporozoite; c, d mononuclear trophozoite; e eight-nuclei meront; f merozoites from eight-nuclei meront; g four-nuclei meront; h merozoites from four-nuclei meront; i and j microgamont; k macrogamont and l zygote. Bar = 10 μm

Fig. 12

Developmental stages of Cryptosporidium chipmunk genotype I in transmission electron microscopy. a Oocyst with four sporozoites (s), residual body (rb) with amylopectin granules (ag), forming oocyst wall (ow) in parasitophorous sac (ps); b early trophozoite with one nucleus (n) inside parasitophorous sac (ps) and attached to microvilli border (mb) with feeding organelle (fo); c later trophozoite with one nucleus (n) inside parasitophorous sac (ps) and attached to microvilli border (mb); d early meront covered with parasitophorous sac (ps) with forming eight merozoites (me) connected to residual body (rb); e cross section of mature meront covered with parasitophorous sac (ps), fully developed eight merozoites (me) with visible nucleus (n) and connected to host cell by feeding organelle (fo); f cross section of early meront with forming four merozoites (me) with visible nucleus (n) and covered with parasitophorous sac (ps); g cross section of mature meront with fully developed four merozoites (me), covered with parasitophorous sac (ps) and attached to host cell by feeding organelle (fo); h empty parasitophorous sac (ps) attached to the host cell by feeding organelle (fo); i released merozoites (me) with visible nucleus (n); j early microgamont (mi) and attached to the host cell by feeding organelle (fo); k macrogamont covered with parasitophorous sac (ps) with foam-like appearance caused by amylopectin granules (ag) and visible nucleus (n); l zygotes with amylopectin granules (3), developing oocyst wall (1), parasitophorous sac (2). Bar = 1 um