Multilocus fragment analysis of Cryptosporidium parvum from pre-weaned calves in Colombia

Abstract

A B S T R A C TThe intra-species genetic diversity of Cryptosporidium parvum in dairy cattle farms in the central area of Colombia was investigated using a multilocus fragment typing approach with nine variable-number tandem-repeat (VNTR) loci and the gp60 gene. Genomic DNA of 70 C. parvum isolates from pre-weaned calves in 32 farms was analysed. Most markers showed two (ML1, MSB, CP47, and MSC6-7) or three alleles (5B12, Cgd2_3850, and Cgd6_5400), although they exhibited a major allele accounting for more than 69% of specimens, which explains their low discriminatory index. The TP14 microsatellite was monomorphic while a total of six alleles were found at the ML2 microsatellite. The two novel allelic variants (219bp, 245bp) exhibited by more than 36% of specimens at the latter locus were a remarkable finding. The 10-markers typing tool provided a Hunter-Gaston discriminatory value of 0.940 (95% CI, 0.918 -0.961) and differentiated 22 multilocus subtypes (MLTs). Nevertheless, the combination of the three most informative markers (ML2, gp60, and Cgd2_3850) differentiated 68% of MLTs and hardly impaired the discriminatory index. The fact that many MLTs (13/22) were distinctive for individual farms provides evidence for the endemic nature of the infection and the major role played by transmission within farms. The eBURST algorithm suggested a low degree of genetic divergence. All but three MLTs were clustered in a clonal complex with a star-like topology typical of clonal expansion, however linkage analysis did not find evidence of linkage disequilibrium. Bayesian analysis also identified a genetic structure with K = 3 being the best estimation of ancestral clusters, although a large proportion of isolates (35%) could not be allocated to a single population, which indicates their mixed origin. The results confirm the genetic distinctiveness of C. parvum in cattle farms in this geographical area. This is the first multilocus analysis on the intra-specific variability of Cryptosporidium from calves in South America.

Multilocus Sequence Typing helps understand the genetic diversity of Cryptosporidium hominis and Cryptosporidium parvum isolated from Colombian patients

“…In our study, this marker was excluded from the unilocus and multilocus analysis since it could not be amplified in all the samples evaluated. Other results obtained in the MLFT study from cattle in Colombia established TP14 as the only marker with a monomorphic behavior [12], which differs from our results, where a polymorphism was identified. However, this could be associated with different analysis strategies and sample origins.…”

“…In Colombia, only one multi-locus analysis work has been published, which was based on the MLFT approach in the study of cattle samples [12]. Avendaño et al found three C. parvum gp60 subtypes that were also identified in our study (IIaA17G4R1, IIaA20G6R1, and IIaA18G5R1), providing evidence of the zoonotic potential of this species and the importance of cattle in cryptosporidiosis transmission in Colombia [12]. In the MLFT analysis, ten genetic markers were included, five of which were also used in our work (CP47, gp60, ML2, MSC6-7, and TP14).…”

“…Studies on Cryptosporidium infection in children and immunocompromised patients in the country report frequencies between 1.9% and 46.8% [7][8][9], and 1% and 51.4% [9][10][11], respectively. Several species have been identified, with most human cases associated with C. hominis and a lower frequency with C. parvum, C. meleagridis, C. felis, Cryptosporidium andersoni, Cryptosporidium ubiquitum, Cryptosporidium muris, Cryptosporidium bovis, and Cryptosporidium viatorum [9,12,13]. In animals and water sources, species such as C. parvum, C. hominis, Cryptosporidium molnari, and Cryptosporidium galli have also been identified [9,12,14].…”