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

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.

Fig 1. Map of Spain showing the three autonomous regions where cattle farms were sampled.

Fig 2. Single-locus variant eBURST network for 70 multilocus subtypes (MLTs) identified among Cryptosporidium parvum isolates from calves.

Dots represent MLTs, with diameters proportional to numbers of isolates. Single locus variants are joined by lines. Distance between dots is random and does not provide additional information. The allelic profile of each MLT is indicated in Table 3.

Fig 3. Population sub-structure based on Bayesian clustering (K = 2) for 102 Cryptosporidium parvum isolates from calves.

Each bar represents an isolate, the colors within the bar reflect the percent assignment (shown on the y-axis) of that specimen to one of two genetic clusters (red or green, respectively). The MLT number for each isolate is shown on the x-axis. Analyses were conducted on allelic data at all VNTR loci and the GP60 marker. The allelic profile of each MLT is indicated in Table 3.