Cryptosporidium species

Carrera-J, Patricio D., ativa, Acosta-Jamett, Gerardo & Munoz ˜, Pamela, 2024, Molecular detection of Cryptosporidium parvum in wild rodents (Phyllotis darwini) inhabiting protected and rural transitional areas in north-central Chile, International Journal for Parasitology: Parasites and Wildlife 24 (4), pp. 123153-123153 : 123153-

3.4. Molecular characterization of Cryptosporidium species and subtypes

Twenty-one (21) PCR products with the highest band intensity in the agarose gel were submitted to Sanger sequencing for the partial 18S rRNA gene. After sequencing, only nine isolates presented homogeneous reads in both directions (i.e., forward and reverse), and the other 12 samples were not adequate for further analysis (ESM 5). After the assemblage and edition of the forward and reverse reads, nine consensus sequences (fragments between 397 and 512 bp) were finally obtained. The BLAST showed the highest homology (between 99% and 100% identity) with various Cryptosporidium parvum isolates.

Maximum likelihood phylogenetic analysis based on an alignment of 466 bp (nucleotide positions 665–1297) of the 18S rRNA gene showed that evaluated consensus sequences of Cryptosporidium found P. darwini in the present study were clustered together within a monophyletic group containing isolates of Cryptosporidium parvum from several host species including ruminants ( China), humans ( Netherlands, Spain), felines ( China), a horse Equus caballus ( Iraq), and river water sample ( Chile) ( Fig. 2 ).

For subtyping, 16 nested PCR products with the highest band intensity in the agarose gel were submitted to Sanger sequencing of partial gp60 gene. After sequencing, seven isolates exhibited both forward and reverse nucleotide sequencing reads. The other nine samples were not appropriate for further analysis. Using the CryptoGenotyper® software ( Yanta et al., 2021), C. parvum subgenotype family IIa was determined in seven samples, including the subtype IIaA17G4R1 (100% identity) in two samples from the protected and rural areas (deposited in GenBank under the accession numbers: PQ084644, PQ084647).

Figures

Fig. 2. Phylogram representing analysis of the 18 rRNA region. The evolutionary history was inferred with maximum likelihood method and the Tamura 3-parameter (T92) model with a discrete Gamma distribution (5 categories (+G)). Analysis contains sequences uploaded from GenBank (with Cryptosporidium species, host, country, and accessions numbers in brackets) and those obtained in the present study are shown in triangles (with ID isolate, host, site of sampling and country, and accessions numbers in brackets). Bootstrap values are represented as per cent of internal branches (1000 replicates), and values lower than 50 are hidden. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Cryptosporidium muris was used to root the tree.