Diversity of Cryptosporidium spp. in Apodemus spp. in Europe

Abstract

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.

Keywords: Epidemiology; Molecular analyses; Phylogeny; Rodentia.

Fig. 1.

Maximum likelihood tree (−ln = 3040.40) based on partial small subunit ribosomal RNA gene sequences of Cryptosporidium, including sequences obtained in this study (highlighted and bolded). The alignment contained 780 base positions in the final dataset. Tamura’s 3- parameter model was applied, using a discrete Gamma distribution and invariant sites. Numbers at the nodes represent the bootstrap values with more than 50% bootstrap support from 1000 pseudoreplicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by isolate number (e.g. 8131), host species (AA for Apodemus agrarius, AF for Apodemus flavicollis and AS for Apodemus sylvaticus) and region (BEL for Belgium, CZE for Czech Republic, FIN for Finland, FRA for France, DEU for Germany, LTA for Latvia, LTU for Lithuania, NLD for Nederland, POL for Poland, ROU for Romania, SRB for Serbia and SVK for Slovakia).

Fig. 2.

Maximum likelihood tree (−ln = 7878.45) based on partial sequences of gene coding actin of Cryptosporidium, including sequences obtained in this study (highlighted and bolded). The alignment contained 695 base positions in the final dataset. The General Time Reversible model was applied, using a discrete Gamma distribution and invariant sites. Numbers at the nodes represent the bootstrap values with more than 50% bootstrap support from 1000 pseudoreplicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by isolate number (e.g. 8131), host species (AA for Apodemus agrarius, AF for Apodemus flavicollis and AS for Apodemus sylvaticus) and region (BEL for Belgium, CZE for Czech Republic, FIN for Finland, FRA for France, DEU for Germany, LTA for Latvia, LTU for Lithuania, NLD for Nederland, POL for Poland, ROU for Romania, SRB for Serbia and SVK for Slovakia).

Fig. 3.

Maximum likelihood tree (-ln = 2652.86) based on partial sequences of gene coding Cryptosporidium oocyst wall protein (COWP), including sequences obtained in this study (highlighted and bolded). The alignment contained 455 base positions in the final dataset. Tamura’s 3- parameter model was applied, using a discrete Gamma distribution and invariant sites. Numbers at the nodes represent the bootstrap values with more than 50% bootstrap support from 1000 pseudoreplicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by isolate number (e.g. 8131), host species (AA for Apodemus agrarius, AF for Apodemus flavicollis and AS for Apodemus sylvaticus) and region (BEL for Belgium, CZE for Czech Republic, FIN for Finland, DEU for Germany, LTU for Lithuania, POL for Poland, ROU for Romania, SRB for Serbia and SVK for Slovakia).

Fig. 4.

Maximum likelihood tree (−ln = 6245.99) based on partial sequences of gene coding 60 kDa glycoprotein of Cryptosporidium (gp60), including sequences obtained in this study (highlighted and bolded). The alignment contained 947 base positions in the final dataset. Tamura’s 3- parameter model was applied, using a discrete Gamma distribution and invariant sites. Numbers at the nodes represent the bootstrap values with more than 50% bootstrap support from 1000 pseudoreplicates. The branch length scale bar, indicating the number of substitutions per site, is given in the tree. Sequences from this study are identified by isolate number (e.g. 8131), host species (AA for Apodemus agrarius, AF for Apodemus flavicollis and AS for Apodemus sylvaticus) and region (CZE for Czech Republic, DEU for Germany, LTA for Latvia, POL for Poland, SRB for Serbia and SVK for Slovakia).