Development and Potential Application of Ras Domain Containing Protein from Haemonchus contortus for Diagnosis of Goat Infection.

Kalibixiati Aimulajiang,Zehua Li,Lixin Xu,Xiaowei Tian,Mingmin Lu,Shakeel Ahmed Lakho,Xiaokai Song,Man Cao,Shuyi Liao,Xiangrui Li,Ruofeng Yan,Muhammad Ali-Ul-Husnain Naqvi

doi:10.3390/ani10010138

Kalibixiati Aimulajiang, Zehua Li + Show 10 more

Open Access

https://doi.org/10.3390/ani10010138

Copy DOI

Abstract

Simple SummaryThe early diagnosis of Haemonchosis is important for the control and effective treatment of the disease. Early stage detection of Haemonchus contortus infection has not been attempted in goats, even though the immature worms are blood-sucking. This study was carried out to detect H. contortus infection during the early and late stages in goats. The results of this study assessed that recombinant H. contortus Ras domain-containing protein (rHcRas) could detect the antibody in H. contortus-infected goat sera during the prepatent and postpatent periods with high sensitivity and specificity using immunodiagnostic techniques. Our findings illustrated that indirect ELISA based on rHcRas also had the potential to detect H. contortus infection from field samples.Haemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide. Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination. However, limitations often occur in early or mild infections. For this purpose, serological diagnosis seems to be more accurate and reliable. Ras domain-containing protein (Ras) is one of H. contortus’s excretory and secretory products (ESPs) that can be isolated from different larval stages of the nematode. In this study, the recombinant H. contortus Ras domain-containing protein (rHcRas) was expressed and purified and its diagnostic potential was evaluated. Reactions between rHcRas and goat sera were tested using Western blotting (WB). The results showed that rHcRas could be recognized by sera as early as 14 days post infection (DPI), and antibodies against rHcRas in infected goats could be maintained for over 89 days. No reaction was found between rHcRas and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immunosorbent assay (ELISA) was produced based on rHcRas. The optimal coating antigen (157 ng of rHcRas/well) and serum dilutions (1:50) were determined via checkerboard titration. Indirect ELISA based on rHcRas showed 87.5% sensitivity and 90.6% specificity. The cut-off values for this experiment were determined to be 0.324 (positive) and 0.273 (negative), respectively, and the variation coefficient (CV) was less than 15%. The results of the indirect ELISA in-field examination showed that 17.6% (9/51) of the goats were infected with H. contortus, higher than the fecal examination results (15.7%, 8/51). When compared the results of the indirect ELISA and necropsy testing, 98.0% (50/51) consistency was found. These results indicated that rHcRas was a potential antigen for the diagnosis of H. contortus infection in goats.

Highlights

Haemonchus contortus is a highly pathogenic, blood-sucking parasite that causes huge production losses of goats worldwide [1]
The positive colonies were grown in Luria Bertini (LB) broth at 37 ◦ C with constant shaking at 120 rpm until an optical density at 600 nm reached 0.6–0.8; the recombinant protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma Aldrich, USA) for 5 h
HcRas was recognized in sera from goats infected with H. contortus, while no protein was detected recombinant HcRas was recognized in sera from goats infected with H. contortus, while no protein in the normal goat sera (Figure 1B)

Summary

Introduction

Haemonchus contortus is a highly pathogenic, blood-sucking parasite that causes huge production losses of goats worldwide [1]. The diagnosis of Haemonchosis is usually based on the detection of clinical signs such as severe anemia. Large numbers of H. contortus in the abomasum verify the presence of the parasite postmortem [1]. Clinical signs appear in the late stage of infection. I.e., loop-mediated isothermal amplification and quantitative polymerase chain reaction, have major significance in enhancing the diagnostic capabilities of Haemonchosis. Their applications are limited to the laboratory, as more sophisticated equipment is required [4,5]. The development of a convenient, rapid, and profitable diagnostic method is needed, especially for the detection of clinical samples

Methods

Results

Discussion

Conclusion