Immunodiagnostic potential of recombinant tropomyosin during prepatent Haemonchus contortus infection in goat

Muhammad Ali-Ul-Husnain Naqvi; Tahseen Jamil; Sana Zahra Naqvi; Muhammad Ali Memon; Kalibixiati Aimulajiang; Muhammad Tahir Aleem; Muhammad Ehsan; Lixin Xu; Xiaokai Song; Xiangrui Li; Ruofeng Yan

doi:10.1016/j.rvsc.2019.11.013

Immunodiagnostic potential of recombinant tropomyosin during prepatent Haemonchus contortus infection in goat

Res Vet Sci. 2020 Feb:128:197-204. doi: 10.1016/j.rvsc.2019.11.013. Epub 2019 Nov 28.

Authors

Affiliations

¹ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2017207047@njau.edu.cn.
² Sindh Agriculture University, Tandojam, Sindh, Pakistan. Electronic address: tahseenjamil1992@gmail.com.
³ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2018207077@njau.edu.cn.
⁴ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2016207040@njau.edu.cn.
⁵ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2017207022@njau.edu.cn.
⁶ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2018207076@njau.edu.
⁷ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: 2014207042@njau.edu.cn.
⁸ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: xulixin@njau.edu.cn.
⁹ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: songxiaokai@njau.edu.cn.
¹⁰ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: lixiangrui@njau.edu.cn.
¹¹ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, China. Electronic address: yanruofeng@njau.edu.cn.

PMID: 31816502
DOI: 10.1016/j.rvsc.2019.11.013

Abstract

Excretory and secretory products (ESPs) are released by the parasites during Haemonchus contortus (H. contortus) infection. In this study, Tropomyosin (TpMy), one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay (ELISA) for detection of H. contortus during early infection in goat. Microscopic examination was performed parallel for comparison. Recombinant tropomyosin protein was purified successfully. Western blotting results revealed that anti-recombinant H. contortus Tropomyosin (rHc-TpMy) antibodies could recognize the natural proteinand rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica, Trichinella spiralis, and Toxoplasma gondii. Moreover, initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection (DPI) and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage (7 and 14 DPI). The optimized antigen coating concentration was calculated as 10 μg/ml (P/N Optimum Density₄₅₀ = 4.165) with optimized dilution of serum (1:50) and secondary antibody (1:2500). Positive and negative cutoff value of the indirect-ELISA assay was calculated as 0.392 and 0.344, respectively. Receiver operating characteristic curve analysis validated the cutoff value (0.392) based on a high specificity and sensitivity. Indirect ELISA showed 90% diagnostic sensitivity and 100% diagnostic specificity. In comparison of serological and conventional method, rHc-TpMy based indirect ELISA showed more positive results (30%; 9/30) than microscopic examination (20%; 6/30). These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.

Keywords: Excretory and secretory products; Haemonchus contortus; Indirect ELISA; Tropomyosin; Western blot.

MeSH terms

Animals
Antibodies, Helminth / blood*
Antigens, Helminth
Blotting, Western / veterinary
Cross Reactions
Enzyme-Linked Immunosorbent Assay / veterinary
Feces / parasitology
Goats
Haemonchiasis / diagnosis*
Haemonchus* / isolation & purification
Haemonchus* / parasitology
Recombinant Proteins / immunology
Sensitivity and Specificity
Serologic Tests / veterinary*
Tropomyosin / immunology*

Substances

Antibodies, Helminth
Antigens, Helminth
Recombinant Proteins
Tropomyosin