Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain

PLoS One. 2016 May 13;11(5):e0155336. doi: 10.1371/journal.pone.0155336. eCollection 2016.

Abstract

A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7-9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979-0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Cryptosporidiosis / parasitology*
  • Cryptosporidium parvum / classification*
  • Cryptosporidium parvum / genetics*
  • Cryptosporidium parvum / isolation & purification
  • DNA, Protozoan
  • Farms*
  • Gene Frequency
  • Genetic Variation*
  • Genetics, Population
  • Geography
  • Linkage Disequilibrium
  • Minisatellite Repeats
  • Multilocus Sequence Typing
  • Sheep
  • Sheep Diseases / epidemiology*
  • Sheep Diseases / parasitology*
  • Spain / epidemiology

Substances

  • DNA, Protozoan

Grants and funding

This work was supported by funds from Spanish (AGL2012-32138) and regional (DGA-B82) research programs, and the European Social Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.