Comparison of Four Diagnostic Methods for Detection and Relative Quantification of Haemonchus contortus Eggs in Feces Samples

Ljungström, Sara; Melville, Lynsey; Skuce, Philip; Höglund, Johan

doi:10.3389/fvets.2017.00239

Cited by 47 publications

(32 citation statements)

References 13 publications

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“…The total egg count was (chamber 1 + chamber 2) multiplied by 100 = EPG. The presence of H. contortus eggs was determined based on their characteristic shape, dark brown blastomeres, and body dimensions (average length = 70 ± 10 µm and width = 45 ± 5 µm) [39,40]. To confirm H. contortus infection for these goats, animals were slaughtered humanely, the worms in the abomasum were checked, and the numbers were counted.…”

Section: Application Of the Indirect Elisamentioning

confidence: 99%

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats

et al. 2019

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Haemonchus contortus, a blood-sucking nematode of ruminants, causes large economic losses worldwide. Diagnosis of infection mainly depends on the evaluation of clinical signs and fecal examination. However, this has limitations for the diagnosis of early or light infections, where serological diagnosis seems to be more accurate and reliable. In this study, the recombinant H. contortus adhesion-regulating molecule protein (rHCADRM) was expressed and purified, and its diagnostic potential was evaluated. Serum samples from goats experimentally infected with H. contortus (n = 5) were collected at 0 (before infection, negative control), 7, 14, 21, 35, 49, 63, 85, and 103 days post-infection (DPI). The reactions between rHcADRM and goat serum were tested using Western blot (WB) analysis. The results show that rHcADRM can be recognized in the serum as early as 14 DPI, and the antibody against rHcADRM in infected goat could be maintained for over 89 days. No reaction was found between rHcADRM and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immune sorbent assay (ELISA) was developed based on rHcADRM. The optimal coating antigen (279 ng of rHcADRM/well) and serum dilutions (1:50) were determined by checkerboard titration. A total of 64 serum samples, including 32 from H. contortus infection goats and 32 from helminth-free goats, were used to determine the positive (0.362) and negative (0.306) cut-off values for the ELISA. The results show this serological diagnosis method is highly sensitive (90.6%) and specific (93.75%). The coefficient of variation within run and between runs was less than 11%. To apply this indirect ELISA during field examination, 51 serum samples were randomly collected from goat farms and tested using this method. The result showed that 19.6% (10/51) of goats were infected with H. contortus, which was 100% consistent with the necropsy result, higher than that of fecal examination (15.7%, 8/51). These results indicate that rHcADRM could be a potential antigen for diagnosis of H. contortus infection in goats.

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Section: Application Of the Indirect Elisamentioning

confidence: 99%

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats

et al. 2019

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“…Moreover, McMaster egg count technique was performed to examine the fecal samples collected at 7, 14, 21, 28 and 35 D.P.I as described previously [50].…”

Section: Methodsmentioning

confidence: 99%

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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“…Fecal samples were collected from all groups at 7, 14, 21, 23, 25, 27, 29, 31 and 35 days postinfection (dpi). McMaster egg count was performed for fecal samples as descripted previously [37]. Additionally, the hatchability percentage of eggs was estimated at 29, 31 and 33 dpi.…”

Section: Infectivity Of Soaked Sirna-2 Treated L3 In Vivomentioning

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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Comparison of Four Diagnostic Methods for Detection and Relative Quantification of Haemonchus contortus Eggs in Feces Samples

Cited by 47 publications

References 13 publications

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection 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

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

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