Introduction
Goat warble fly infestation (GWFI) is a myiasis caused by larvae ofPrzhevalskiana silenus (P. silenus) which belongs to subfamilyHypoderminae of Oestridae family1. This disease is associated with huge economic losses by reduction in milk yield, meat and hide quality2, 3, and it is characterized by the presence of subcutaneous warbles on dorso-lumbar region of affected goats and wild ruminants.4 All three larval stages (L1, L2 and L3) cause subcutaneous infestation for 7-9 months and no internal migration is observed in contrast to other hypoderminae members such as Hypoderma bovis (H. bovis ) and Hypoderma lineatum (H. lineatum ).5
GWFI is widespread and commonly present in the Mediterranean Basin and Indian subcontinent that includes northern India, Pakistan, Iran, Turkey and southern Italy.2,6-10 In India, the disease is most common in northwestern Himalayan area (Union Territory of Jammu and Kashmir), particularly in goats of the Bakerwali breed characterized by long hairy coat with a prevalence rate ranging between 13 to 56.5 % in Jammu region.2
Physicoclinical observation by palpation of warbles on infested animals is routinely used for diagnosis at L2 and L3 stages which occurs about 3 months after infestation. Serological tests have advantages over physicoclinical observation in terms of early detection and high sample throughput along with wide diagnostic time window, as antibodies in the infested host may persist for about 16 weeks.11,12 The serodiagnostic assays for hypoderminae insects have been explored with the key serine proteases namely Hypodermin A (HyA), Hypodermin B (HyB) and Hypodermin C (HyC). Among the three serine proteases, HyC is primarily produced in early stage of L1 that helps in the penetration of the skin due to its collagenolytic activity. HyC is characterized as a major immunodominant antigen. The conserved nature of HyC shows cross reactivity among Hypoderminae subfamily due to shared epitopes.11, 13-18
Several enzyme immunoassays have been developed based on native and recombinant HyC derived from H. bovis and H. lineatumbased indirect ELISAs have been developed for diagnosis of cattle hypodermosis.19-23 Presently, recombinant protein based microtitre plate-ELISAs are widely used in routine diagnosis and seroepidemiological studies of hypodermosis.6,18,23However, the assay is expensive, time-consuming and requires equipment like ELISA reader and expertise to analyse the data. Diagnostic labs performing spectrophotometric ELISA are scarce in developing countries, especially at remote veterinary diagnostic centres. In contrast to such challenges, a simple, rapid and easy to perform test is required. Dot-ELISA is an inexpensive, sensitive and specific assay that provides utility to be performed at less equipped regional labs and provide visual readouts without spectrophotometry.24
Several dot-ELISA have been developed for various zoonotic and veterinary parasitic diseases such as Toxocariosis based onToxocara canis Excretory –Secretory Antigens (TES), Taenia solium cysticercosis (neurocysticercosis) with partially purified antigen fraction (Cathepsin L like activity); Toxoplasmosis based on recombinant surface antigen (rSAG) and dense granule antigens (rGRA7), recombinant cathepsin L-cysteine proteinase for Fasciola gigantica , truncated recombinant merozoite antigen (rBgSA1) forBabesia gibsoni among others.24-28
The present study aimed to develop a dot-ELISA based on recombinant HyC derived from P. silenus for the serodiagnosis of GWFI in order to develop an economical, rapid and field applicable tool for serosurveillance of this myiasis during disease control programs.