Integrated epidemiological and molecular analysis of Cryptosporidium spp. and Giardia duodenalis isolates in dairy calves from Terceira Island, Azores

Cryptosporidium spp. and Giardia duodenalis are major enteric pathogens in neonatal calves, contributing to gastrointestinal disease and raising concerns due to their zoonotic potential. Despite their global distribution, little is known about their occurrence and genetic diversity in insular regions such as the Azores archipelago (Portugal). This study investigated the prevalence, distribution, genetic diversity, and farm-level risk factors associated with these parasites in dairy calves from Terceira Island. Fecal samples (n = 142) from 28 farms were examined by microscopy, direct immunofluorescence assay (DFA), and PCR targeting multiple loci (SSU rRNA, gp60, LSU rRNA for Cryptosporidium spp.; gdh, bg, tpi for G. duodenalis). Sequencing enabled molecular identification of species, genotypes, and assemblages. Farm management data were collected by standardized questionnaires, and spatial analysis used farm location. Overall, 65.5% (93/142) of calves were infected with at least one parasite, with Cryptosporidium spp. and Giardia spp. detected in 42.3% (60/142) and 44.4% (63/142) of samples, respectively. Mixed infections occurred in 21.1% (30/142) of calves. Molecular characterization identified C. parvum (subtype IIaA15G2R1), C. bovis, and C. ryanae, as well as G. duodenalis assemblage E (predominant) and assemblage A (rare). Spatial mapping showed Cryptosporidium spp. to be widespread, while Giardia spp. displayed localized clusters. Use of paromomycin and higher hygiene scores were associated with reduced Giardia risk, whereas no risk factors were found for Cryptosporidium. These findings provide novel insights into protozoan infections in calves in the Azores and highlight the value of integrating molecular, spatial, and management data for targeted control strategies.