title: Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain.
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.
authors.affiliation: Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Ramo
authors.affiliation: Department of Anatomy, Embryology and Genetics, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain.
authors.forename: Luis V
authors.initials: LV
authors.lastname: Monteagudo
authors.affiliation: Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Del Cacho
authors.affiliation: Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain.
authors.forename: Caridad
authors.initials: C
authors.lastname: Sánchez-Acedo
authors.affiliation: Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Spain.
authors.forename: Joaquín
authors.identifier: 0000-0001-7831-2483
authors.initials: J
authors.lastname: Quílez
mesh_terms: D000483:Alleles; D000818:Animals; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D016054:DNA, Protozoan; D000072480:Farms; D005787:Gene Frequency; D014644:Genetic Variation; D005828:Genetics, Population; D005843:Geography; D015810:Linkage Disequilibrium; D018598:Minisatellite Repeats; D058885:Multilocus Sequence Typing; D012756:Sheep; D012757:Sheep Diseases / Q000453:epidemiology* / Q000469:parasitology*; D013030:Spain / Q000453:epidemiology
chemical_list: D016054:DNA, Protozoan