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.