doi:10.1371/journal.pone.0155336engRamo, A.Monteagudo, L.V.Del Cacho, E.Sánchez-Acedo, C.Quílez, J.Intra-species genetic diversity and clonal structure of Cryptosporidium parvum in sheep farms in a confined geographical area in northeastern SpainART-2016-94807A 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.2016http://zaguan.unizar.es/record/4869310.1371/journal.pone.0155336http://zaguan.unizar.es/record/48693oai:zaguan.unizar.es:48693info:eu-repo/grantAgreement/ES/DGA/B82info:eu-repo/grantAgreement/ES/MINECO/AGL2012-32138PloS one 11, 5 (2016), 0155336 [16 p.]byhttp://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccess