DEVELOPMENT AND EVALUATION OF RECOMBINANT PROTEIN BASED ELISA FOR THE DIAGNOSIS OF INTESTINAL SCHISTOSOMOSIS
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Date
2019-12-30
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
Animal schistosomosis caused by Schistosoma spindale is an
economically considerable blood fluke infection that adversely affects the
livestock sector in India. The aim of the present study was to clone, express,
purify and analyse 22.6 kDa tegument protein of S. spindale (rSs22.6 kDa) in
prokaryotic system and to assess its usefulness as a diagnostic candidate for
seroprevalence studies. An abattoir survey of intestinal schistosomosis in
bovines conducted in Thrissur corporation slaughter house, Kuriachira from
August, 2017 to July, 2018 revealed an overall prevalence of 25 per cent of
intestinal schistosomosis in cattle with 24.34 per cent of S. spindale and 0.66
per cent of S. indicum infection. The highest prevalence of infection (26.32
per cent) was observed during monsoon season. Majority of the samples
showed low intensity infections (81.57 per cent). Occurrence of the disease or
the intensity of the infection related with seasons did not show any statistical
significance. For the production of recombinant 22.6 kDa protein, RNA was
isolated from adult live schistosome worms, cDNA synthesized and the
specified 22.6 kDa tegument protein coding gene was amplified and cloned in
pJET cloning vector and transformed in E. coli Top 10 cells. The confirmed
product (573bp) was amplified with primers having restriction site for BbS1
and digested with BbS1 restriction enzymes. Then pET28b expression vector
was digested with Xho1 and NCo1 restriction enzymes and transformed to
BL21 E. coli cells. Induction of protein was done 0.6mM IPTG for four
hours. Purification of the soluble fraction of newly expressed polyhistidine
(6X-His) tagged fusion protein was carried out by Nickel chelating affinity
chromatography using Ni-NTA agarose column. The SDS PAGE analysis of
rSs22.6 protein and the following Coomassie Brilliant Blue staining of the gel
revealed a bright single band of size approximately 22.6 kDa. Moreover,
immunoblotting of rSs22.6 protein with schistosome positive bovine serum
revealed a single immunodominant protein corresponding to the approximate
molecular weight of 22.6 kDa without any cross reaction with amphistome
positive bovine serum. Standardisation of IgM and IgG based Dot and indirect ELISA was carried with rSs22.6 protein. Excretory secretory antigens
(ESA) were also prepared using the mesentery recovered adult schistosomes
to conduct ESA based indirect IgG ELISA. Copro PCR was carried out with
mitochondrial gene specific primers of Schistosoma spp. and an amplicon of
approximately 454 bp size was amplified indicating the presence of S.
spindale. Comparison of results of rSs22.6 IgM ELISA with that of copro
PCR revealed a sensitivity of 16.67 per cent while that of copro PCR was 30
per cent with a cent percent specificity. The results showed that the sensitivity
of rSs22.6 IgM ELISA was lower than that of copro PCR, suggesting
unsuitability of rSs22.6 IgM indirect ELISA for seroprevalence studies. The
diagnostic performance of rSs22.6 IgG ELISA was compared with ESA IgG
ELISA and copro PCR using 38 known positive samples and 13 known
negative samples. A sensitivity of 92.11 per cent for rSs22.6 IgG ELISA, 89.47 per cent for ESA IgG ELISA and 31.58 per cent for copro PCR was
observed whereas specificity was cent per cent for all the three assays.
Sensitivity of rSs22.6 IgG ELISA higher than ESA IgG ELISA. Sensitivity of
copro PCR was considerably low. However, there was no statistically
significant difference between the sensitivities of rSs22.6 IgG ELISA and
ESA IgG ELISA while both these assays showed statistically significant
difference from copro PCR. Seroprevalence study with rSs22.6 IgG ELISA of
506 sera samples of cattle from southern, central and northern zones of
Kerala revealed a prevalence status of 26.88 per cent of intestinal
schistosomosis. Highest prevalence of the infection was observed in
Alappuzha district (42.86 per cent) and coastal sandy zone (41.18 per cent)
whereas the lowest prevalence was in Ernakulam (17.19 per cent) district and
Red loam (15.63 per cent) zone of Kerala without any statistically significant
difference between these findings.
In silico analysis of the expressed protein revealed that it is a protein with 190
amino acids. Predicted secondary structure of protein revealed the presence of
alpha helices (47.89 per cent), extended strands (17.37 per cent), beta turns (8.95
per cent) and random coils (25.79 per cent). The tertiary structure of the protein
revealed that it contained α helices and β sheets with EF-hand as a helix-loop- helix domain. Analysis of rSs22.6 protein showed that signal peptides were
absent. Presence of transmembrane helices in the predicted protein sequence
indicated the absence of transmembrane helices. The NCBI conserved domain
prediction revealed the presence of two conserved domains, one EF-hand domain
located in its N-terminus (residues 12–71) and a dynein light-chain domain
located in its C-terminus (residues 99–186). The possible number and composition
of epitopes were predicted by linear epitope prediction in Immune Epitope
Database and Analysis Resource tool revealed the presence of seven epitopes with
aminoacid sequences ranging from 3 to 20. Phylogenetic analysis using the
Maximum Likelihood method in MEGA 5.2. revealed that it was a sister clade of
S. haematobium and S. bovis while it is distinct from the clade containing S.
japonicum.
Description
Submitted in partial fulfilment of the requirement for the degree of
Doctor of Philosophy in Veterinary Parasitology