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.