Development of a Quantitative Real-Time PCR Assay for Detection of Cryptosporidium spp. Infection and Threatening Caused by Cryptosporidium parvum Subtype IIdA19G1 in Diarrhea Calves from Northeastern China

Parasitic diarrheal disease is a major cause of morbidity and mortality in the developing world. Calves are highly susceptible to Cryptosporidium spp. infection that resulted in diarrhea, growth retardation, and weight loss, and was one of the most common enteropathogens. It is especially difficult for molecular detection of calves with inapparent or subclinical infections of cryptosporidiosis. In view of this, this study established a real-time quantitative PCR (RT-qPCR) detection method to clarify its epidemic characteristics, based on Cryptosporidium 18S rRNA gene with the 150 bp product length to investigate the infection of Cryptosporidium spp. in northeastern China The standard curve equation is Ct = -2.91 × lg (Cryptosporidium spp. copies) +10.18, with better sensitivity, stability, and reproducibility. A total of 148 out of 425 fecal samples (34.82%) were detected Cryptosporidium positive with RT-qPCR, including (36.11%) in Heilongjiang province (29.60%), (29.6%) in Jilin province, and (37.50%) in Liaoning province. The infection prevalence of Cryptosporidium parvum, Cryptosporidium ryanae, Cryptosporidium andersoni, and Cryptosporidium bovis from calves in order from high to low was 14.35% (95% confidence interval [CI], 11.2-18.1), 6.12 (95% CI, 4.0-8.8), 2.35 (95% CI, 1.1-4.3), and 0.47 (95% CI, 0.1-1.7), respectively, suggesting C. parvum was the predominant species in calves in northeastern China. Using 60-kDa glycoprotein gp60 gene, all of the 61 C. parvum-positive specimens were further precisely confirmed to IIdA19G1 subtype. This suggested that IIdA19G1 subtype of C. parvum could threaten to cause diarrhea calves from notheastern China (p < 0.01). The prevalence of 34.82% (148/425) using RT-qPCR had a significant difference compared with the prevalence of nested-PCR (23.29%) and microscopic examination (3.76%). The findings improved the epidemiological knowledge of calves infected with cryptosporidiosis in China, highlighting the importance of ongoing Cryptosporidium surveillance.