Discussion
Goat warble fly infestation is an economically important disease of
goats. In India, the disease is prevalent in Union Territory of Jammu
and Kashmir (North India)2 and causes huge economic
losses in the region.3 Globally, GWFI causes huge
production losses to goat farming in the Mediterranean and Indian
subcontinent with consistent reports from south Italy, Greece, Egypt,
Jordan, Saudi Arabia, Iraq, Iran, Pakistan with variable prevalence
rates over the years.4 The development of a sensitive
and specific technique to diagnose warble fly infestation is an
important step towards promoting sustainable control of the
disease.30,31
The diagnosis of GWFI is commonly based on the physicoclinical
observation of subcutaneous warbles at the late stage of L2 and L3 which
occur after 3-4 months of infestation. For early and confirmatory
diagnosis, serological test has been developed for the detection ofHypoderma spp. specific antibodies. Many workers have developed
crude and native HyC based indirect ELISA derived from H.
lineatum with DSn 100%, DSp 92%;21 DSn 98.2% and
DSp 98.2%,20 respectively. However, it is well known
that crude or native HyC antigen preparation is challenging to
standardize and availability of L1 larvae throughout the year is not
feasible because of the seasonal pattern of the disease and variable
prevalence in a geographical region. An alternative approach is the use
of recombinant protein produced in heterologous system instead of native
antigen. This provides the advantage of purified specific antigen and
amenability to standardization of immunoassay with less background
signal. Recombinant protein based immunoassays overcomes the limitations
associated with available serodiagnostic tests. HyC is the major
immunodominant protein that is present in all members ofHypoderminae . Indirect ELISA based on the rHyC of H.
lineatum 22,20 and H. bovis 23have been developed and used successfully for detecting
hypoderma-specific antibodies from cattle serum.
A competitive ELISA based on crude lysate protein derived fromPrzhevalskiana spp. has been developed for the early detection of
GWFI in goats (8). Microtitre plate ELISA has also been developed based
on P. silenus derived recombinant HyC.18However, the serosurveillance based on microtitre plate ELISA should be
performed by skilled personnel in spectrophotometry equipped
laboratories which is impractical at field laboratory in the developing
countries in much of the Indian subcontinent and Mediterranean Basin.
Field applicability of diagnostic tests is a desirable attribute for
livestock farmers with small holdings in developing countries which can
provide sensitive and specific diagnosis. Upon testing the dot-ELISA
within a limited trial at ambient temperature (18 °C) the results were
found to be valid and supported the suitability of the test under field
conditions for the diagnosis of GWFI under sub-tropical and temperate
zones. Also, the time required to perform the present dot-ELISA after
blocking of strips is about 1 h 15 min which further supports the field
applicability. Several other studies on parasitic diagnosis based on
dot-ELISA require a time period ranging between 1h 35 min to 3 h after
blocking step in various assays for other parasitic
diseases.25,26,32 The shorter incubation time and
temperature conditions makes the assay amenable to field application.
As described earlier, diversity of antigenic preparations have been used
for parasite immunodiagnosis with dot-ELISA such as excretory-secretory
antigens, crude lysate preparations, purified native
antigens.25,26 However, in the recent decades,
recombinantly expressed protein antigens have been emphasized through
various studies for their immunodiagnostic
applications.24,27,28,32
In the present study, dot-ELISA was standardized based on rHyC derived
from P .silenus for serodiagnosis of GWFI. The expression of rHyC
fusion protein derived from P. silenus in prokaryotic expression
system has been previously optimized.18 The rHyC
fusion protein was produced in bulk and purified under denaturing
condition and predicted size of purified fusion protein
~45 kDa was obtained. The yield of expressed protein
after dialysis was about 3 mg/L of induced culture which is sufficient
to screen approximately 5000 serum samples in triplicate dots.
Moreover, recombinant antigen source provides a sustainable and
consistent source of antigen for diagnostic application all-round the
year thus eliminating the need of larval origin antigens, apart from
providing a diagnostic test with improved parameters. The purified rHyC
protein reacted very well with GWFI positive serum in dot-ELISA format
at an antigen concentration of 188 ng/dot and antibody and conjugate
dilution 1:800 and 1:4000 respectively. The diagnostic sensitivity and
diagnostic specificity of rHyC dot-ELISA were found to be 97.3 % and
95.8 %, respectively. The sensitivity and specificity of the developed
dot-ELISA were found comparable as to those of rHyC based
ELISA.20,22,23 Recombinant antigen used as purified
antigen helps in reduction of non-specific reaction.33The assessment at lower time and temperature conditions for incubation
showed variable intensity of dots. This variation in dot color intensity
might be attributed to the prozone phenomenon as neat samples might
present disproportionately high amount of antibodies in the GWFI
positive samples.34 Samples with weaker signals were
taken as positive and the dot ELISA was applied for qualitative
assessment of 274 samples for GWFI. The dot-ELISA optimized in the
present study provides a high serum dilution rate at 1:800 which permits
the scope of using pooled serum samples for the assessment of herd
screening for warble fly infestation.31,35 The present
dot ELISA provides a high sample dilution rate which improves
specificity and may be used with dried blood sample on filter paper
availed in field conditions with minimal resources for GWFI diagnosis
(36). This provides an opportunity for a standardized simple diagnostic
tool for mass surveillance of GWFI at the national eradication program.
The optimized dot-ELISA was found to be highly specific with GWFI serum
and did not exhibit any visible reactivity with other important
parasitic and bacterial diseases of goats. The rHyC based dot-ELISA was
found to be non-reactive to Oestrus ovis positive sera of goats
which is in accordance with the previous observation in western blotting
and microtitre rHyC indirect-ELISA.14,18 The optimized
dot-ELISA can serve as economical, efficient, field applicable and thus
forms a suitable tool for serosurveillance of GWFI during disease
control program.
Moreover, blotting technique including dot-ELISA has multiple advantages
over indirect ELISA since protein adsorption is higher in Nitrocellulose
membranes,37, 38 which permits the higher dilution of
samples as observed in the optimized assay. The technique provides
simple execution and interpretation with minimal reagents. Also, it can
be used for large volume tests or small number of
samples.39, 40
The cross-reactivity of HyC due to its conserved nature and shared
epitopes among Hypoderminae members has been evaluated in several
studies.11, 13, 14, 16, 41, 42 This suggests that the
rHyC based dot-ELISA can be used for serodiagnosis of hypodermosis in
cattle, yalk and cervids with other hypoderminae infestation such asH. sinense, H. bovis, H. lineatum, H. diana and H.
actaeon . Antibody kinetics studies for hypoderminae infestation show
that antibodies are observed as early as fourth week of L1 infestation
and persists up to 16 to 20 weeks.11 This provides an
opportunity for early diagnosis based on antibody detection and
intervention at L1 stage with prophylactic treatment to avoid production
losses to milk, meat and hide.11
The antigen dotted membranes can be stored for up to 6 months at both 4
°C and 37 °C without any changes in
reactivity.27,36,43 This stability of NCM coated
antigen dots makes it more suitable for field application with minimal
storage specification at regional veterinary centers. Further
thermostability studies of antigen dotted NCM strips would be required
for longer time period of storage.