Cryptosporidium myocastoris n. sp. (Apicomplexa: Cryptosporidiidae), the Species Adapted to the Nutria (Myocastor coypus)

Abstract

Cryptosporidium spp., common parasites of vertebrates, remain poorly studied in wildlife. This study describes the novel Cryptosporidium species adapted to nutrias (Myocastor coypus). A total of 150 faecal samples of feral nutria were collected from locations in the Czech Republic and Slovakia and examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, and gp60 loci. Molecular analyses revealed the presence of C. parvum (n = 1), C. ubiquitum subtype family XIId (n = 5) and Cryptosporidium myocastoris n. sp. XXIIa (n = 2), and XXIIb (n = 3). Only nutrias positive for C. myocastoris shed microscopically detectable oocysts, which measured 4.8-5.2 × 4.7-5.0 µm, and oocysts were infectious for experimentally infected nutrias with a prepatent period of 5-6 days, although not for mice, gerbils, or chickens. The infection was localised in jejunum and ileum without observable macroscopic changes. The microvilli adjacent to attached stages responded by elongating. Clinical signs were not observed in naturally or experimentally infected nutrias. Phylogenetic analyses at SSU, actin, and HSP70 loci demonstrated that C. myocastoris n. sp. is distinct from other valid Cryptosporidium species.

Keywords: adaptation; biology; course of infection; infectivity; oocyst size; parasite; phylogeny; prevalence.

Figure 1

Sampling localities in the Czech Republic and Slovakia. For each site, the number indicates the name of locations (1) Planá nad Lužnicí (N 49°21.11527′, E 14°42.08325′); (2) Praha (N 50°4.78070′, E 14°24.81330′); (3) Jihlava (N 49°23.09785′, E 15°36.37422′); (4) Třebíč (N 49°12.58360′, E 15°52.28832′); (5) Břeclav (N 48°46.34147′, E 16°52.68835′); (6) Týnec (N 48°46.31570′, E 17°0.66778′); (7) Lanžhot (N 48°43.41558′, E 16°58.30782′); (8) Nové Zámky (N 48°0.76540′, E 18°11.75573′); (9) Komárno (N 47°45.04053′, E 18°8.98083′); (10) Šaľa (N 48°9.08273′, E 17°52.49152′); (11) Doný Ohaj (N 48°4.33237′, E 18°14.81102′); (12) Topolníky (N 47°57.61112′, E 17°45.37918′); (13) Palárikovo (N 48°2.15190′, E 18°2.70193′); (14) Nitrianský Hrádok (N 48°3.62700′, E 18°12.56517′); (15) Dunajská Streda (N 47°55.90470′, E 17°28.42662′); (16) Vlčny (N 48°2.69967′, E 17°57.72202′); (17) Diakovce (N 48°8.02725′, E 17°50.48138′); and (18) Lipové (N 47°50.41113′, E 17°51.33057′). The colour indicates the presence of Cryptosporidium spp.

Figure 2

Maximum likelihood tree based on partial small subunit ribosomal RNA gene sequences of Cryptosporidium spp., including sequences obtained in this study (bolded and highlighted). The alignment contained 770 base positions in the final dataset. Numbers at the nodes represent the boot strap values with more than 50% boot strap support from 1000 pseudo replicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by an isolate number (e.g., 32247). Black circles and squares indicate natural and experimental infections, respectively.

Figure 3

Maximum likelihood tree based on actin gene sequences of Cryptosporidium spp., including sequences obtained in this study (bolded and highlighted). The alignment contained 990 base positions in the final dataset. Numbers at the nodes represent the boot strap values with more than 50% boot strap support from 1000 pseudo replicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by an isolate number (e.g., 32247). Black circles and squares indicate natural and experimental infections, respectively.

Figure 4

Maximum likelihood tree based on 70 kDa heat shock protein (HSP70) gene sequences of Cryptosporidium spp., including sequences obtained in this study (bolded and highlighted). The alignment contained 1172 base positions in the final dataset. Numbers at the nodes represent the boot strap values with more than 50% boot strap support from 1000 pseudo replicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by an isolate number (e.g., 32247). Black circles and squares indicate natural and experimental infections, respectively.

Figure 5

Maximum likelihood tree based on 60 kDa glycoprotein (gp60) gene sequences of Cryptosporidium spp., including sequences obtained in this study (bolded and highlighted). The alignment contained 1206 base positions in the final dataset. Numbers at the nodes represent the boot strap values with more than 50% boot strap support from 1000 pseudo replicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by an isolate number (e.g., 32247). Black circles and squares indicate natural and experimental infections, respectively.

Figure 6

Course of infection of Cryptosporidium myocastoris n. sp. in experimentally inoculated nutria (Myocastor coypu). (A) Infection intensity expressed as number of oocysts per gram of faeces (OPG), and (B) detection of oocysts is based on molecular and microscopic examinations of faecal samples. Black squares indicate the presence of oocysts and specific Cryptosporidium myocastoris n. sp.; grey squares indicate the detection of specific DNA only without oocyst detection. Hatched rectangles indicate a missing animal due to sacrifice and dissection.

Figure 7

Histological sections stained by periodic acid–Schiff showing developmental stages of Cryptosporidium myocastoris n. sp. (arrow) on (a) jejunal and (b,c) ileal mucosal epithelium in experimentally infected adult nutria (Myocastor coypu) which was sacrificed 10 days post infection. Scale bar is included in each figure.

Figure 8

Scanning electron microphotograph showing developmental stages of Cryptosporidium myocastoris n. sp. (arrow) on (a) jejunal and (b) ileal mucosal epithelium in experimentally infected adult nutria (Myocastor coypu) which was sacrificed 10 days post infection. Elongation of the microvilli around attached developmental stage (arrowhead). Scale bar included in each figure.

Figure 9

Oocysts of Cryptosporidium myocastoris n. sp. (a) in differential interference contrast microscopy, (b) stained by aniline–carbol–methyl violet staining, (c) stained by Ziehl–Nielsen staining, and (d) labelled with anti-Cryptosporidium FITC-conjugated antibody. Bar = 5 μm.