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

Naqvi, Muhammad Ali-ul-Husnain; Jamil, Tahseen; Naqvi, Sana Zahra; Memon, Muhammad Ali; Aimulajiang, Kalibixiati; Aleem, Muhammad Tahir; Ehsan, Muhammad; Xu, Lixin; Song, Xiaoling; Li, Xiangrui

doi:10.1016/j.rvsc.2019.11.013

Cited by 15 publications

(12 citation statements)

References 33 publications

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“…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.…”

Section: Discussionsupporting

confidence: 88%

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

et al. 2020

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Background: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 μg/well at 37°C 1 h and overnight at 4°C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD 450 ). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

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Section: Discussionsupporting

confidence: 88%

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

et al. 2020

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“…The suppressive activity was further noted with a decreased level of IL-4 and IFN-γ production in addition to downregulation of the chemical factor NO, cell proliferative efficiency and cell migratory activity, which ultimately increased apoptosis of goat PBMCs. In our recent study, TpMy was found to be an important indicator with high immunodiagnostic potential for early diagnosis of H. contortus infections [22]. However, further study is urgently needed to check the effectiveness of rHc-TpMy in the goat-infected model, which could help understand profoundly the molecular mechanism(s) of this protein during host-parasite interactions.…”

Section: Discussionmentioning

confidence: 97%

“…In our previous research, tropomyosin was identified as an excretory and secretory protein (ESP) that binds to goat PBMCs at the L4 and L5 developmental stages of H. contortus [20], and we also demonstrated that HcESPs exhibit suppressive regulatory roles in their interaction with PBMCs [21]. In our recent study, recombinant tropomyosin was found to have high immunodiagnostic potential for early stage diagnosis of H. contortus infection in goats [22]. However, the immunological functional impacts of Hc-TpMy on goat PBMCs remained unknown.…”

Section: Introductionmentioning

confidence: 91%

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Ehsan

Haseeb

et al. 2020

Vaccines

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During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

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“…Western blotting played a preliminary role in the selection of the target protein to evaluate the immunogenicity and immuno-reactivity of the antigen [32]. Antigenic characteristics of rHc-MMP-12 were also evaluated in sera of goats infected with H. contortus as described previously [33]. First, purified rHc-MMP-12 was separated on 12% SDS-PAGE and a semi dry system (NovablotHoefer, San Francisco, USA) was used to transfer onto PVDF (polyvinyl difluoride membrane; Millipore, Billerica, USA) with transfer solution (Tris 48 mM, glycine 39 mM, SDS 0.0375%, methanol 20%).…”

Section: Western Blot Assaymentioning

confidence: 99%

Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity

Naqvi

Gao

et al. 2020

Parasites Vectors

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Background: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized.Methods: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat.Results: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNAtreated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. Conclusions:Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

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

Cited by 15 publications

References 33 publications

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

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

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro

Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity

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