Prevalence, molecular characterization, and risk factor analysis of Cryptosporidium parvum in dairy calves from Punjab, India.

Cryptosporidium infection is a well-established, widely recognized pathogen affecting neonatal dairy calves worldwide, with profound implications for both animal welfare and public health. This investigation evaluated the prevalence, molecular characteristics, and risk determinants of Cryptosporidium infection across seven districts of the Central Plain Zone (CPZ) of Punjab, India. A total of 341 fecal samples from dairy calves (180 buffalo and 161 cattle) were analyzed using modified Ziehl-Neelsen (mZN) staining of direct fecal smears (DFS) and saturated sugar flotation smears (SSFS), the latter employed to enhance oocyst concentration and improve detection sensitivity. Microscopic examination revealed an overall prevalence of 23.75% (81/341), with 39 samples testing positive by DFS and 81 by SSFS, establishing SSFS as the superior diagnostic method. Geographic analysis demonstrated significant spatial heterogeneity, with Ludhiana district exhibiting the highest prevalence (38.46%, P < 0.05) and Shri Fatehgarh Sahib the lowest (6.25%). Multifactorial risk analysis identified four critical determinants of infection: periurban location (38.46%, P < 0.01), age < 1 month (32.4%, P < 0.01), diarrheic status (29.59%, P < 0.01), and organized farm management systems (28.72%, P < 0.05). Molecular characterization through nested PCR amplification of the 18S rRNA gene (∼834 bp) followed by PCR-RFLP analysis using restriction enzymes (SspI, VspI, and MboII) conclusively identified Cryptosporidium parvum as the sole species present in all positive samples from both cattle and buffalo calves. BLASTn analysis showed 98.86% and 98.61% homology with GenBank C. parvum sequences for cattle and buffalo isolates, respectively. Phylogenetic analysis revealed that Punjab isolates clustered with C. parvum strains from diverse geographic regions, including other Indian states (Uttar Pradesh, Bihar) and international locations (Algeria, Bangladesh, China), while remaining distinct from C. bovis, C. ryanae, C. andersoni, and C. hominis. The exclusive detection of C. parvum, based on the few samples analysis, needs further gp60 subtyping to delineate circulating IIa genotypes on more numbers of samples in remaining zones of the Punjab for exploring the zoonotic potential as risk posed by infected dairy calves and underscores the urgent need for comprehensive surveillance and control measures accordingly.