Comparative evaluation of various diagnostic techniques for detection of Cryptosporidium infection from the faecal samples of diarrhoeic bovine calves

Background: Cryptosporidium, an opportunistic, zoonotic, apicomplexan parasite, is one of the most common causes of diarrhea in neonatal bovine calves around the globe. Bovine calves act as a major source of infection by excreting huge numbers of highly resistant oocysts in faeces, which can survive for a long time in extreme environmental conditions. As low as ten oocysts can cause disease and mortality, leading to the requirement of an early and accurate diagnosis for proper and favorable prognosis, management, and control. Aims: The current study was conducted with the objective to evaluate various diagnostic techniques (acid fast staining, negative staining, fluorescent, ELISA, PCR, nested PCR, and qPCR) for the detection of Cryptosporidium in the faecal samples of diarrheic bovine calves. Methods: Two hundred diarrheic faecal samples from bovine calves were collected and subjected to these techniques for Cryptosporidium diagnosis. Results of these were evaluated for diagnostic comparison. Results: Out of 200 faecal samples evaluated, 24% (48/200) were detected positive for Cryptosporidium using a combination of two techniques as gold standard criteria. Cohen's kappa value indicated moderate to almost perfect agreement (0.616 to 0.986) among all the techniques used in the present study. Leishman staining showed the lowest sensitivity (54.17%), while nested PCR and qPCR showed the highest sensitivity (97.92%). Diagnostic specificity of all these tests ranged from 98.68 to 100%. Conclusion: Auramine stain was used for the first time in the bovine calves in India for the detection and diagnostic comparison of Cryptosporidium. It showed strong agreement with the molecular as well as classical diagnostic techniques, and can be used for primary screening for better diagnosis.