Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Abstract

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

“…In present study, both sensitivity and specificity of the rHc-CS indirect-ELISA were measured as 100%, that is similar to the findings of previous study in which His-ES24-based ELISA was performed to detect specific antibodies in H. contortus infected sera of sheep [17]. In contrast, 90% [43] and 87% [41] diagnostic sensitivity of indirect ELISA was reported using different recombinant H. contortus tropomyosin antigens.…”

“…Indirect ELISA based on rHc-CS was optimized and developed to complement the immunoblotting results. Previously, combined use of ELISA and immunoblotting has been reported as a powerful tool to immuno-diagnose different infections [38,41]. In this study, rHc-CS indirect-ELISA showed higher diagnostic specificity and sensitivity as compared to somatic antigen-based indirect-ELISA format reported in previous study [20].…”

“…It helps to select target protein for the diagnostic purposes as well as for the antigenic immunogenicity and immunoreactivity evaluation [52]. Immunoblotting was performed to evaluate the immunoreactivity of rHc-CS using rat sera following the methods as described in our previous study [41]. Moreover, immunoblotting analysis was also performed to evaluate the antigenic characteristics of rHc-CS at early stage as well as late stage of H. contortus infection in sera of experimentally infected goats (Group 1).…”

“…In order to evaluate the diagnostic potential and to complement the immunoblotting results, standardized indirect-ELISA was developed as described previously [41,55]. Moreover, serum samples (n = 33) from infected goats were used to calculate diagnostic sensitivity and serum samples of all goats before artificial infection (n = 35) were used to calculate diagnostic specificity of rHc-CS based indirect ELISA using following formula [56].…”

Immunomodulatory dynamics of excretory and secretory products on Th9 immune response during Haemonchus contortus infection in goat

“…Another study also reported transgenic expression of IL-9 in mice infected with Trichuris muris and Trichinella spiralis [16,17]. Respectively, Haemonchus contortus is the most important gastrointestinal nematode parasite which is responsible for brutal health problems and economic losses in the small ruminant industry worldwide [18,19]. It is a bloodsucking parasite that feeds on blood, results in anemia, dehydration, loss of body weight and even death in young animals [20].…”