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

Ana Ramo,Joaquín Quílez,Emilio Del Cacho,Caridad Sánchez-Acedo,Luis V Monteagudo

doi:10.1371/journal.pone.0155336

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.

Highlights

Cryptosporidium parvum is an ubiquitous and significant entero-pathogen causing diarrheal illness in many species of mammals, humans and young livestock
The percentage of specimens allocated to each allelic variant revealed that over 93% of isolates shared the same allele at CP47 and MSC6-7 loci, which explains the low discriminatory index for both markers
Five isolates showed a biallelic profile at 5B12, GP60, MSB, Cgd3_3850 or Cgd6_5400 loci, which evidenced the presence of intra-host mixed infections

Summary

Introduction

Cryptosporidium parvum is an ubiquitous and significant entero-pathogen causing diarrheal illness in many species of mammals, humans and young livestock. Human-specific, animal-specific and zoonotic subtypes have been identified using sequence analysis of the 60 kDa glycoprotein (GP60) gene, which is the single most polymorphic marker identified so far in the Cryptosporidium genome. C. parvum is the most prevalent species in diarrheic pre-weaned lambs in most European countries [3,4,5,6], but modest numbers of ovine specimens have been genetically characterized using GP60 sequencing [6,7,8,9,10,11], and other VNTR loci [7, 12,13,14,15,16,17]. All ovine isolates have been found to belong to the potentially zoonotic subtype families IIa and IId, and both sporadic and outbreak-related human cases involving direct contact with lambs have been documented [18,19]

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Conclusion