Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 10⁰⁴ and 5.95 × 10⁰⁵ , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.