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.

Muhammad Ali-Ul-Husnain Naqvi,Kalibixiati Aimulajiang,Xiangrui Li,Lixin Xu,Ruofeng Yan,Muhammad Ali Memon,Muhammad Haseeb,Sana Zahra Naqvi,Xiaokai Song

doi:10.3390/ani9080548

Muhammad Ali-Ul-Husnain Naqvi, Kalibixiati Aimulajiang + Show 7 more

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https://doi.org/10.3390/ani9080548

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Abstract

Simple SummaryThe accurate and early diagnosis of Haemonchus contortus infection is crucial for effective control. Early stage detection of H. contortus infection has not been attempted in goat, even though both immature worm and fourth larval stage are blood sucking. This study was carried out to detect the H. contortus infection during early stage in goat. The results of this study assessed that rHc-HCA59 could detect the antibody in H. contortus infected goats’ sera during early period with good sensitivity and specificity using immunodiagnostic techniques. Our findings illustrated that combined use of ELISA and western blotting based on rHc-HCA59 is a powerful tool for early detection of H. contortus infection in goats.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.

Highlights

Small ruminant farming industry contributes one of the major means to develop living standards in numerous developing countries and plays an important role in the national economy [1]
The results of this study concluded that combined use of indirect enzyme linked immunosorbent assay (ELISA) and western blotting with rHc-Hepatocellular carcinoma-associated antigen 59 (HCA59) is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats
The same band was observed in the Western blotting and confirmed the presence of band that corresponded to the protein of interest (Figure 1B)

Summary

Introduction

Small ruminant farming industry contributes one of the major means to develop living standards in numerous developing countries and plays an important role in the national economy [1]. Goats are vulnerable to gastrointestinal nematodes in humid, tropical, and subtropical regions and are overwhelmed by Haemonchus contortus (H. contortus) [2]. It is an important blood sucking parasite found in abomasum that may remove about 0.05 mL of blood per day by seepage from lesions [3]. Prepatent stage detection of H. contortus infection has not been attempted in goats, even though both immature worm and fourth larval stage are blood sucking. It is difficult to detect H. contortus eggs in feces before the third week of post infection (21–25 days) [5]. Until the infection shows clinical signs, young animals suffer from anemia, diarrhea, edema, weight loss, severe frailty, and death [6]

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