Molecular characterization of Cryptosporidium isolates from high-excreting young dairy calves in dairy cattle herds in Western France

Ninety-two Cryptosporidium sp.-positive fecal samples of dairy diarrheic or non-diarrheic calves from 30 cattle herds in Normandy (France) were selected. Here, the aim was to investigate the species of Cryptosporidium excreted as well as the subtypes of Cryptosporidium parvum found in 7-17-day-old dairy calves. Excretion levels were comprised between 2 x 10(4) and 4 x 10(7) oocysts per gram of feces. Here, a nested 18S SSU rRNA PCR associated with sequencing was performed for identification of Cryptosporidium species and revealed the presence of C. parvum in most cases (80/82), except for two animals which were infected with Cryptosporidium bovis. Then, C. parvum samples were submitted to gp60 PCR. For 39 samples from 24 different herds, a multilocus analysis based on four mini-microsatellites loci (MM19, MM5, MSF, and MS9-Mallon) were conducted. These results were combined with sequence analysis of the gp60 to obtain multilocus types (MLTs). Here, C. parvum gp60 genotyping identified three subtypes in the IIa zoonotic allele family: IIaA15G2R1 (88 %), IIaA16G3R1 (10 %), and IIaA19G2R1 (2 %), and we identified 12 MLTs. The MS9-Mallon locus was reported as the most polymorphic (five alleles). The most common MLT was MLT 1 with 15 samples in 10 farms: (MS9-M: 298, MSF: 165, MM5: 264, MM19: 462, and gp60 subtype: IIaA15G2R1). When comparing diarrheic and non-diarrheic fecal samples, no difference was seen for distribution of Cryptosporidium species, C. parvum gp60 subtypes, and MLTs. Here, in a range of oocyst excretion of 10(4)-10(7) opg, both in diarrheic and non-diarrheic calves, infection was mainly due to C. parvum and to the zoonotic subtype: IIaA15G2R1.