Calf diarrhea is a multifactorial disease that poses substantial challenges to the cattle industry, necessitating timely identification and characterization of causative agents to inform effective control strategies. This study investigated the prevalence, co-occurrence, and characteristics of Cryptosporidium and Eimeria infections in diarrheic calves. A total of 348 diarrheal fecal samples were collected from calves aged 1–30 days across 15 prefectures in Japan between February 2020 and February 2022. Preliminary diagnosis of calf diarrhea was performed using an immunochromatographic test (ICT) kit. Cryptosporidium spp. were detected in 36.5% (127/348) of samples. Using nested PCR and conventional PCR with species-specific primers, targeting these two major parasitic pathogens, we assessed seasonal variations, prevalence, and associations with calf age, breed, and sex. Overall, Cryptosporidium was detected in 58.6% of samples, while Eimeria spp. were found in 7.5%. Mixed infections involving both pathogens occurred in 4.9% of samples. The highest detection rates for both pathogens were observed in calves aged 8–15 days, with a significant age-related decline in mixed infections (p < 0.05). Seasonal variations were observed, with distinct prevalence patterns for each pathogen. Breed- and sex-related differences were minimal but are described in detail. Phylogenetic analysis confirmed C. parvum as the predominant Cryptosporidium species and E. bovis as the most common Eimeria species. These findings highlight the widespread presence of Cryptosporidium and Eimeria spp. in diarrheic calves in Japan and provide insights into age- and season-related infection dynamics, informing targeted control and management strategies.
