Intra-Species Diversity and Panmictic Structure of Cryptosporidium parvum Populations in Cattle Farms in Northern Spain.

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

doi:10.1371/journal.pone.0148811

Abstract

The intra-herd and intra-host genetic variability of 123 Cryptosporidium parvum isolates was investigated using a multilocus fragment typing approach with eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene. Isolates were collected from intensively farmed diarrheic pre-weaned calves originating from 31 dairy farms in three adjoining regions in northern Spain (País Vasco, Cantabria and Asturias). The multilocus tool demonstrated an acceptable typeability, with 104/123 samples amplifying at all twelve loci. The ML2, TP14, GP60 and the previously un-described minisatellite at locus cgd2_3850 were the most discriminatory markers, while others may be dismissed as monomorphic (MSB) or less informative (CP47, ML1 and the novel minisatellites at loci Cgd1_3670 and Cgd6_3940). The 12-satellite typing tool provided a Hunter-Gaston index (HGDI) of 0.987 (95% CI, 0.982–0.992), and differentiated a total of 70 multilocus subtypes (MLTs). The inclusion of only the four most discriminatory markers dramatically reduced the number of MLTs (n: 44) but hardly reduced the HGDI value. A total of 54 MLTs were distinctive for individual farms, indicating that cryptosporidiosis is an endemic condition on most cattle farms. However, a high rate of mixed infections was detected, suggesting frequent meiotic recombination. Namely, multiple MLTs were seen in most farms where several specimens were analyzed (90.5%), with up to 9 MLTs being found on one farm, and individual specimens with mixed populations being reported on 11/29 farms. Bayesian Structure analysis showed that over 35% of isolates had mixed ancestry and analysis of evolutionary descent using the eBURST algorithm detected a high rate (21.4%) of MLTs appearing as singletons, indicating a high degree of genetic divergence. Linkage analysis found evidence of linkage equilibrium and an overall panmictic structure within the C. parvum population in this discrete geographical area.

Highlights

Cryptosporidium parvum is a prevalent and clinically important pathogen causing gastrointestinal disease in a variety of vertebrate hosts, humans and neonatal domestic ruminants
It is worth mentioning the finding of previously un-described alleles at three loci and the high variability of the novel locus Cgd3_3850, which seems a good candidate for future multilocus analyses
The 12-loci typing method provided a high discriminatory power, but the exclusion of the three less polymorphic markers (MSB, CP47, Cgd1_3670) hardly reduced the number of multilocus subtypes (MLTs), and the inclusion of only four markers (ML2, TP14, GP60 and Cgd3_3850) hardly impaired the Hunter-Gaston discriminatory index (HGDI) value, indicating a redundancy which has been detected in most multilocus schemes [28]

Summary

Introduction

Cryptosporidium parvum is a prevalent and clinically important pathogen causing gastrointestinal disease in a variety of vertebrate hosts, humans and neonatal domestic ruminants. The use of generation sequencing applications and other tools such as automated fragment analysis have revealed an unexpectedly high intra-isolate genetic heterogeneity, with important implications for our understanding of cryptosporidiosis epidemiology [8,9,10]. All these data highlight the need of validated tools to unravel the population genetic structure and elucidate the transmission pathways of C. parvum. Three different and opposing genetic structures have been proposed for C. parvum, from a panmictic population in which sexual recombination is frequent, to a clonal theory, and an epidemic population structure where there is a background level of frequent sexual recombination with the occasional clonal expansion of a few particular haplotypes [11]

Methods

Results

Conclusion