1. INTRODUCTION
Cryptosporidium is an intestinal protozoan parasite that infects a wide range of livestock and vertebrate hosts[1,2]. It is an opportunistic pathogen responsible for gastrointestinal disease, especially in the immune-compromised host[3]). Cryptosporidium infection is also responsible for mortality and morbidity in severely affected animals and less than five years of children lead to diarrhoea, weakness, weight loss and late maturity, reported 526,000 deaths annually worldwide in 2015 [4]. Cryptosporidium was detected worldwide in young sheep and goats in diarrheic and non-diarrheic conditions with a higher rate of prevalence. Cryptosporidiuminfection has been reported highest in Spain (62.7%) and Mexico (72.5%) in goat kids [5,6].
The oocysts of various Cryptosporidium species infecting humans and livestock are not readily distinguishable based on morphological characteristics by the conventional diagnostics or microscopic examination [7]. Moreover, microscopy-based methods require skilled personnel and are labour-intensive[8]. Nowadays, several molecular techniques have been developed to overcome this problem and increasing as first-line diagnosis procedures for the identification of Cryptosporidiuminfection in faecal samples [9,10]. A wide range of polymerase chain reaction (PCR) assays are available with higher sensitivity and specificity including simplex PCR[11-13], multiplex real-time PCR and duplex qRT-PCR for detecting Cryptosporidium spp. with the stage of infection and differentiating the live and dead oocysts[14]. Molecular assays are highly sensitive to the purity and quality of DNA material.
However, DNA extraction by conventional method is a multi-step procedure by using various reagents and incubation steps. TheCryptosporidium oocysts consist of three layers of filamentous glycoprotein (oocyst wall protein (OWP)) and acid-fast lipids. COWP play a major structural and functional role in Cryptosporidiumoocysts. The structural make-upof COWP comprises of two different amino acid motifs: Type I and Type II repeatspresent with six cysteine residues and the higher amount of glycine and proline residues. The structural arrangement of COWP consist two major domains: amino-terminal domain and carboxyl-terminal domain. The signal peptide (extracellular localization)of COWP consist 1622 amino acids, first 698 amino acids consist in ten Type I repeats of approximately 65 amino each and 772 amino acids consist in eight Type II repeats of approximately 53 amino acids. This complex structure of Cryptosporidium oocysts wall haywires the DNA extraction by conventional methods[15-18]. So there is dire necessity for an effective DNA extraction method that could effectively break the thick-walled oocyst of Cryptosporidium spp. and remove the PCR inhibitors from the faecal samples [19-26]. Also PCR based downstream analysis (PCR-RFLP, sequencing and phylogenetic analysis) offers better understanding in the disease dynamics compared to microscopy, while the latter also cannot assess the type of strains and sub-types of Cryptosporidium species in a particular geo-climatic zone. DNA extraction is the key bottleneck that needs to be addressed for a successful geo-epidemiological disease dynamics and effective management of cryptosporidiosis.
Hence in the current study, to overcome these constraints, a new conventional DNA extraction protocol was developed and evaluated for rapid screening ofCryptosporidium spp. in faecal samples by conventional and real-time PCR. The aim of this study is also to compare the current method with commercially available kits for DNA extraction in terms of quality, quantity and workability in PCR based tests.