No abstract

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 × 10 04 and 5.95 × 10 05 , 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.dead and live oocysts, duplex real-time PCR assay, oocyst wall protein | INTRODUCTIONCryptosporidiosis disease is caused by the intracellular, extracytoplasmic protozoan parasite, which belongs to the phylum Apicomplexa. Cryptosporidiosis is the primary cause of chronic diarrhea among immunocompromised patients, especially in children suffering from HIV, AIDS-defining illness because of its high association with mortality. [1][2][3][4] Cryptosporidium is accountable for 8%-19% of diarrhea in less developed countries, 5 and till the year 2010, it already caused about 100,000 deaths globally. 6 More than 15% of the world's population is drinking contaminated water and suffering from waterborne parasitic protozoan diseases globally, 7 it is responsible for four billion cases of mild to severe diarrhea and 1.6 million deaths annually. 8,9 Cryptosporidium oocysts are

DNA extraction from stools is the major hurdle in the detection of Cryptosporidium infection due to complex oocyst wall and presence of PCR inhibitors. There is no conventional full-proof DNA extraction method which efficiently recovers DNA from Cryptosporidium. Alternatively, commercial DNA kits costs dearer and unaffordable in smaller laboratories with limited funding. To address this, the current study explored for an efficient DNA extraction method from Cryptosporidium oocysts. Four different DNA-extraction methodologies were developed at different Cetyltrimethylammonium Bromideconcentrations and temperature-cycles and analyzed for quality-quantity parameters. Four different types of recipes were used, in brief which includes 1. Sonication+3 cycles of chill-thaw+ (24:1) Chloroform:Isoamyl alcohol, 2. Liquid Nitrogen+ thaw (@70ºC)+ (24:1) Chloroform: Isoamyl alcohol, 3. Sonication + 3 cycles of freeze that + (24:1) Chloroform: Isoamyl alcohol and 4. Three cycles of ‘snap-chill (@-80ºC) and boil (@95ºC) +(24:1) Chloroform: Isoamyl alcohol’. Among these, a protocol involving three cycles of ‘snap-chill and boil’ (Method-4) could successfully recover Cryptosporidium DNA with better quality-quantity parameters with consistency and repeatability and lack of PCR inhibitors as evidenced by the workability of this method confirmed by conventional-PCR and real-time PCR for 18 small-subunit-ribosomal RNA ( 18SSU rRNA). The repeated deep freezer and boil cycles successfully disrupted the thick chitin-rich oocyst wall of Cryptosporidium leading to precipitation of nucleic acids by chloroform-isoamyl alcohol. The current study aims to introduce a cost-effective method that overcomes the bottlenecks faced with the conventional DNA extraction techniques for Cryptosporidium directly from faecal samples.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhoea 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. For COWP and 18SSU rRNA genes, the limit of detection was 7.08x1004 and 5.95x1005 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 the healthy animals.