First report of Przhevalskiana silenus derived recombinant hypodermin C based indirect ELISA for serodiagnosis of goat warble fly myiasis

Yadav, Anish; Rafiqi, Shafiya Imtiaz; Yadav, Vikas; Kushwaha, Anand; Godara, Rajesh; Sood, Shilpa; Bhat, M. Iqbal Jeelani; Katoch, Rajesh; Panadero-Fontán, Rosario

doi:10.1038/s41598-022-17760-5

Cited by 3 publications

(21 citation statements)

References 45 publications

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“…Microtitre plate ELISA has also been developed based on P. silenus derived recombinant HyC. 18 However, the serosurveillance based on microtitre plate ELISA should be performed by skilled personnel in spectrophotometry equipped laboratories which is impractical at field laboratory in the developing countries in much of the Indian subcontinent and Mediterranean Basin. Field applicability of diagnostic tests is a desirable attribute for livestock farmers with small holdings in developing countries which can provide sensitive and specific diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…The recombinant HyC protein was derived from P. silenus expressed in Escherichia coli (strain BL21 DE3 pLysS) transformed with the pET-32a(+) expression vector as described in Yadav et al 18 The rHyC was obtained as fusion protein with thioredoxin tag and used as antigen in the present study. Briefly, a confirmed positive recombinant clone was cultured in LB broth containing ampicillin and chloramphenicol, incubated at 37 o C till it attains an OD of 0.6.…”

Section: Recombinant Hyc Antigenmentioning

confidence: 99%

“…Also, the inter-rater reliability of dot-ELISA was evaluated with Cohen's Kappa test, taking the rHyC based microtitre plate indirect-ELISA as reference standard. 18,29 A set of 149 sera samples (69 positive and 80 negative) from ICAR-NF laboratory were tested for evaluating the kappa index between both assays for the diagnosis of GWFI.…”

Section: Evaluation Of Rhyc Based Dot-elisamentioning

confidence: 99%

“…The conserved nature of HyC shows cross reactivity among Hypoderminae subfamily due to shared epitopes. 11,[13][14][15][16][17][18] Several enzyme immunoassays have been developed based on native and recombinant HyC derived from H. bovis and H. lineatumbased indirect ELISAs have been developed for diagnosis of cattle hypodermosis. [19][20][21][22][23] Presently, recombinant protein based microtitre plate-ELISAs are widely used in routine diagnosis and seroepidemiological studies of hypodermosis.…”

Section: Introductionmentioning

confidence: 99%

“…[19][20][21][22][23] Presently, recombinant protein based microtitre plate-ELISAs are widely used in routine diagnosis and seroepidemiological studies of hypodermosis. 6,18,23 However, the assay is expensive, time-consuming and requires equipment like ELISA reader and expertise to analyse the data. Diagnostic labs performing spectrophotometric ELISA are scarce in developing countries, especially at remote veterinary diagnostic centres.…”

Section: Introductionmentioning

confidence: 99%

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Dot-ELISA based on recombinant Hypodermin C protein derived from Przhevalskiana silenus for field diagnosis of goat warb

Yadav

Rafiqi

et al. 2023

Preprint

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Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing prolonged economic loss to animal productivity. The early diagnostic intervention is needed during the disease monitoring and prophylactic management for effective control of the disease. The present study has developed an in-house dot-ELISA for the serodiagnosis of GWFI based on recombinant Hypodermin C (rHyC) antigen of Przhevalskiana silenus, expressed in E. coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen’s kappa index of 0.973. Further testing at ambient temperature (18 C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The rHyC protein based Dot-ELISA was evaluated using random field serum samples suspected for GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective tool for sustainable control of GWFI.

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Section: Discussionmentioning

confidence: 99%

Section: Recombinant Hyc Antigenmentioning

confidence: 99%

Section: Evaluation Of Rhyc Based Dot-elisamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Dot-ELISA based on recombinant Hypodermin C protein derived from Przhevalskiana silenus for field diagnosis of goat warb

Yadav

Rafiqi

et al. 2023

Preprint

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Dot‐ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation

Yadav,

Rafiqi,

Yadav

et al. 2023

Parasite Immunology

Self Cite

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Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot‐ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter‐rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti‐goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter‐rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot‐ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.

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Comparative Analysis of Different ELISA Methods for the Serodiagnosis of Przhevalskiana silenus Infestation in Goats

Liaqat

Qayyum

Çelik

et al. 2023

Veterinary Sciences

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Przhevalskiana silenus (warble fly) grubs cause myiasis in goats, in mountainous and semi-mountainous areas and different regions in Pakistan, and cause substantial losses to livestock. The palpation method for detecting warble flies generally neglects the infestation intensity; therefore, the development of a reliable and efficient diagnostic technique is extremely necessary. This study compared three indirect enzyme-linked immunosorbent assay (ELISA) methods for detecting anti-P. silenus antibodies using the hypodermin C (HyC) purified from Hypoderma spp. Larvae collected in cattle (local isolate, Microbiology Laboratory, PMAS-Arid Agriculture University, Rawalpindi), the crude antigen from the first instar stage of P. silenus, and a commercial Bovine Hypodermosis Antibody ELISA kit (IDEXX Laboratory), for accurately estimating the seroprevalence of goat warble fly infestation (GWFI) in the Pothwar plateau, Punjab, Pakistan. The ELISA with the crude antigen of P. silenus proved very sensitive and specific, 91% and 93%, respectively. The optical density exhibited a monthly variation, and the antibody titer began increasing from June, continually increased from July to December, and gradually decreased thereafter until March. The study confirmed the endemic status of GWFI in the Pothwar region and identified that ELISA based on the crude antigen of P. silenus was a more sensitive and specific immunodiagnostic method for determining seroprevalence, and could be employed for initiating nationwide eradication campaigns.

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First report of Przhevalskiana silenus derived recombinant hypodermin C based indirect ELISA for serodiagnosis of goat warble fly myiasis

Cited by 3 publications

References 45 publications

Dot-ELISA based on recombinant Hypodermin C protein derived from Przhevalskiana silenus for field diagnosis of goat warb

Dot-ELISA based on recombinant Hypodermin C protein derived from Przhevalskiana silenus for field diagnosis of goat warb

Dot‐ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation

Comparative Analysis of Different ELISA Methods for the Serodiagnosis of Przhevalskiana silenus Infestation in Goats

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