First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves

Abstract

Background: The genetic characterization of Cryptosporidium and Giardia has important implications for investigating their epidemiology and underpins their control. We undertook the first molecular epidemiological survey of domestic bovids in selected regions of Sri Lanka to establish whether they excreted Cryptosporidium and/or Giardia with zoonotic potential.

Methods: Faecal samples were collected from dairy calves (n = 340; Bos taurus; < 3 months of age; weekly sampling for six weeks) and water buffaloes (n = 297; Bubalus bubalis; <6 months and ≥6 months of age; one sampling) from seven different farms in Sri Lanka. Genomic DNAs were extracted from individual faecal samples and then tested for the presence of parasite DNA using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing genetic markers within the small subunit of nuclear ribosomal RNA and 60 kDa glycoprotein genes (designated pSSU and pgp60, respectively) for Cryptosporidium, and within the triose phosphate isomerise (ptpi) gene for Giardia.

Results: Based on pSSU sequence data, C. bovis, C. ryanae and six new genotypes that were genetically similar but not identical to C. andersoni (n = 1), C. bovis (n = 1), C. ryanae (n = 3) and C. suis (n = 1) were recorded in cattle. For pSSU, two other, new genotypes were defined in water buffalo, which were genetically most similar to Cryptosporidium genotypes recorded previously in this host species in other countries including Australia. Consistent with the findings for pSSU, no species or genotypes of Cryptosporidium with zoonotic potential were detected using pgp60. Based on ptpi sequence data, G. duodenalis assemblages A and E were detected in four and 137 samples from cattle, respectively, and assemblage E in two samples from water buffaloes.

Conclusions: The present study showed that C. parvum, the most commonly reported zoonotic species of Cryptosporidium recognised in bovine calves globally, was not detected in any of the samples from pre-weaned calves tested in the present study. However, eight new genotypes were recorded. Future studies of different host species in various regions are required to investigate the molecular epidemiology of cryptosporidiosis and giardiasis in Sri Lanka and neighbouring countries in South Asia.

Figure 1

Phylogenetic analysis of pSSU sequence data for Cryptosporidium using Bayesian inference (BI) analysis. The ten distinct pSSU sequences determined in the present study and 36 reference sequences representing Cryptosporidium (accession nos. listed in Additional file 1) were included in the analysis. Eimeria necatrix, E. acervulina, E. brunetti, E. praecox and E. maxima sequences were used as outgroups. Accession numbers of publicly available sequences are indicated at the ends of the branches. Accession numbers linked to sequences determined in the present study are in bold-type; the numbers of samples with particular sequence types are in parentheses. Posterior probabilities (pp) for major nodes are indicated.

Figure 2

Phylogenetic relationships of ptpi sequences of Giardia duodenalis based on Bayesian inference (BI) analysis. Sixteen sequences determined in the present study, and 41 reference sequences representing G. duodenalis assemblages A to G (accession nos. listed in Additional file 4) were included in the analysis. Sequences representing G. ardeae, G. muris and G. microti were used as outgroups. Accession numbers linked to sequences determined in the present study are in bold-type; the numbers of samples with particular sequence types are in parentheses. Posterior probabilities (pp) are indicated at all major nodes.