\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Dot-ELISA based on recombinant Hypodermin C protein of Przhevalskiana
silenus for field diagnosis of goat warble fly infestation}



\author[1]{Vikas Yadav}%
\author[1]{Shafiya Imtiaz Rafiqi}%
\author[1]{Anish Yadav}%
\author[1]{Anand Kushwaha}%
\author[1]{Rajesh Godara}%
\author[1]{Rajesh Katoch}%
\author[2]{Rosario Panadero‑Fontán}%
\affil[1]{Sher-e-Kashmir University of Agricultural Sciences and Technology Faculty of Veterinary Sciences and Animal Husbandry RS Pura}%
\affil[2]{Universidade de Santiago de Compostela - Campus de Lugo}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Goat warble fly infestation (GWFI) is an economically important myiasis
caused by larvae of \emph{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
\emph{Przhevalskiana silenus,} expressed in \emph{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
\textsuperscript{o}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.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Goat warble fly infestation (GWFI) is a myiasis caused by larvae
of\emph{Przhevalskiana silenus (P. silenus)} which belongs to
subfamily\emph{Hypoderminae} of Oestridae family\textsuperscript{1}.
This disease is associated with huge economic losses by reduction in
milk yield, meat and hide quality\textsuperscript{2, 3}, and it is
characterized by the presence of subcutaneous warbles on dorso-lumbar
region of affected goats and wild ruminants.\textsuperscript{4} All
three larval stages (L1, L2 and L3) cause subcutaneous infestation for
7-9 months and no internal migration is observed in contrast to other
hypoderminae members such as \emph{Hypoderma bovis} (\emph{H. bovis} )
and \emph{Hypoderma lineatum} (\emph{H. lineatum} ).\textsuperscript{5}

GWFI is widespread and commonly present in the Mediterranean Basin and
Indian subcontinent that includes northern India, Pakistan, Iran, Turkey
and southern Italy.\textsuperscript{2,6-10} In India, the disease is
most common in northwestern Himalayan area (Union Territory of Jammu and
Kashmir), particularly in goats of the Bakerwali breed characterized by
long hairy coat with a prevalence rate ranging between 13 to 56.5 \% in
Jammu region.\textsuperscript{2}

Physicoclinical observation by palpation of warbles on infested animals
is routinely used for diagnosis at L2 and L3 stages which occurs about 3
months after infestation. Serological tests have advantages over
physicoclinical observation in terms of early detection and high sample
throughput along with wide diagnostic time window, as antibodies in the
infested host may persist for about 16 weeks.\textsuperscript{11,12} The
serodiagnostic assays for hypoderminae insects have been explored with
the key serine proteases namely Hypodermin A (HyA), Hypodermin B (HyB)
and Hypodermin C (HyC). Among the three serine proteases, HyC is
primarily produced in early stage of L1 that helps in the penetration of
the skin due to its collagenolytic activity. HyC is characterized as a
major immunodominant antigen. The conserved nature of HyC shows cross
reactivity among Hypoderminae subfamily due to shared
epitopes.\textsuperscript{11, 13-18}

Several enzyme immunoassays have been developed based on native and
recombinant HyC derived from \emph{H. bovis} and \emph{H. lineatum}based
indirect ELISAs have been developed for diagnosis of cattle
hypodermosis.\textsuperscript{19-23} Presently, recombinant protein
based microtitre plate-ELISAs are widely used in routine diagnosis and
seroepidemiological studies of
hypodermosis.\textsuperscript{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. In contrast to such challenges, a simple, rapid and
easy to perform test is required. Dot-ELISA is an inexpensive, sensitive
and specific assay that provides utility to be performed at less
equipped regional labs and provide visual readouts without
spectrophotometry.\textsuperscript{24}

Several dot-ELISA have been developed for various zoonotic and
veterinary parasitic diseases such as Toxocariosis based
on\emph{Toxocara canis} Excretory --Secretory Antigens (TES),
\emph{Taenia solium} cysticercosis (neurocysticercosis) with partially
purified antigen fraction (Cathepsin L like activity); Toxoplasmosis
based on recombinant surface antigen (rSAG) and dense granule antigens
(rGRA7), recombinant cathepsin L-cysteine proteinase for \emph{Fasciola
gigantica} , truncated recombinant merozoite antigen (rBgSA1)
for\emph{Babesia gibsoni} among others.\textsuperscript{24-28}

The present study aimed to develop a dot-ELISA based on recombinant HyC
derived from \emph{P. silenus} for the serodiagnosis of GWFI in order to
develop an economical, rapid and field applicable tool for
serosurveillance of this myiasis during disease control programs.

\textbf{Material \& methods}

\textbf{Recombinant HyC antigen}

The recombinant HyC protein was derived from \emph{P. silenus} expressed
in \emph{Escherichia coli} (strain BL21 DE3 pLysS) transformed with the
pET-32a(+) expression vector as described in Yadav et
al.\textsuperscript{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\textsuperscript{o}C till
it attains an OD of 0.6. Subsequently it was induced by adding IPTG to a
final concentration of 1 mM. The culture was allowed to grow at
37\textsuperscript{o}C with shaking at 200 rpm for 6 h and purified
under denaturing condition using 8 M urea dialyzed against gradient urea
concentration (8 M to 0 M) and finally in Tris buffer pH 8.0. The
homogeneity of purified protein was analyzed by 12\% SDS-PAGE and
quantified by Bradford reagent.

\textbf{Reference Sera}

Positive reference goat serum samples (n=109) were collected from farms
and a municipal slaughterhouse from Union Territory of Jammu and Kashmir
(North India), based on palpation of larvae in live animals and carcass
examination. Negative goat sera (n=92) were procured from Punjab state
of North India, where GWFI has never been reported. In order to rule out
the cross reactivity of developed dot-ELISA, positive serum of several
important parasitic and bacterial diseases of goats namely oestrosis,
haemonchosis,fasciolosis, amphistomiosis, coenurosis, cryptosporidiosis,
paratuberculosis, enterotoxaemia and brucellosis were assessed.

\textbf{Optimization of rHyC based Dot-ELISA}

Dot ELISA for simple and rapid diagnosis of GWFI was standardized on
strips of Nitrocellulose membrane (NCM) with pore size of 0.45 \selectlanguage{greek}µ\selectlanguage{english}m using
positive and negative reference serum samples for GWFI. NCM were cut
into 0.8 cm x 2.5 cm strips and rHyC protein was dotted at three
different places on to membrane maintaining the gap of 0.5 cm to allow
separation between the dots. The working concentration of recombinant
antigen, serum antibodies and conjugate for dot-ELISA were determined by
standard checkerboard titration. Two different concentrations of
purified rHyC antigen 94 ng/dot (1 \selectlanguage{greek}µ\selectlanguage{english}L) and

188 ng/dot (2 \selectlanguage{greek}µ\selectlanguage{english}L) were titrated against three dilutions of serum (1:400,
1:800 and 1:1200) and two dilutions (1:4000 and 1:8000) of anti-goat IgG
HRP conjugate in dot ELISA format using 5\% skimmed milk powder (SMP) in
PBS as blocking buffer. The antigen concentration, serum and conjugate
dilution that showed the development of well-defined dots on the
membrane with positive serum and none with negative serum were selected.
The optimal conditions included NCM strips dotted with rHyC proteins 2
\selectlanguage{greek}µ\selectlanguage{english}L dots. The dotted strips were air dried for 15 min at room temperature
and blocked with 5\% SMP in PBS (pH 7.4). After incubation at 37 \selectlanguage{ngerman}°C for
1 h, strips were washed two times with PBS-T 0.075\% and one time with
PBS. After washing, strips were soaked in 1 mL of known positive and
negative serum sample diluted in PBS (1:800) and incubated at 37 °C for
1 h. After wash, the strips were incubated with 1 mL of anti-goat IgG
HRP-conjugate (Sigma Aldrich, USA) at 1:4000 dilution in PBS (pH 7.4) at
37 °C for 1 h. Following three washing, ELISA dots were developed by
incubating the strips with DAB substrate (VWR-Amresco Life Science, 5
mg/10 mL) solution for 5 min at room temp in dark. The reaction was
stopped by washing the strips in distilled water. The development of
brown dots on strips indicated that serum samples were positive for GWFI
while negative sera showed no dots. All the strips were handled with
forceps.

\textbf{Evaluation of rHyC based dot-ELISA}

The dot ELISA was performed with rHyC under optimized conditions to
determine the assay specificity and cross reactivity using known
positive sera against different important parasitic and bacterial
diseases of goats. The diagnostic specificity (DSp) and diagnostic
sensitivity (DSn) of the dot ELISA were determined by, using a two-sided
contingency table \textbf{(Table 1.)} . Known positive serum samples
with brown spots compared to nil in negative serum were reported as
positive. The sensitivity and specificity were calculated by using the
following formula: Sensitivity = (TP/TP+FN) × 100; Specificity =
(TN/TN+FP) × 100; where, TP-true positive; TN-true negative; FP-false
positive and FN-false negative. The positive predictive value (PPV) and
negative predictive value (NPV) were also calculated based on the
optimized assay as shown in \textbf{Table 1} .

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.\textsuperscript{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.

In a separate experiment, the optimized dot ELISA was tested with neat
samples (blood, plasma and serum) and lower dilutions at lower
incubation temperature (18 °C) and time (30 min) to check the
suitability of the test at field conditions under sub-tropical and
temperate climates of GWFI prevalent regions. Samples of a known GWFI
positive goat were used for blood and plasma whereas a GWFI negative
goat was used to derive negative control samples of blood, plasma and
serum. The positive samples of GWFI (blood, plasma and weak positive
serum) were tested as neat, 1:2 and 1:10 dilutions and control negative
samples for neat blood, plasma and serum. A weak positive serum was
taken as positive control to ensure reactivity of weak titre at lower
time and temperature of incubation.

Serum samples (n = 274) from different regions of Union territory of
Jammu and Kashmir that were available in the ICAR-NF laboratory were
tested with the optimized dot-ELISA.

\textbf{Stability of developed Dot-ELISA strips}

The stability of antigen dotted NCM strips was assessed at different
time and temperature combinations with a panel of GWFI positive and
negative sera. Strips were dotted with a fixed concentration of rHyC
antigen (188 ng/dot), blocked with 5\% SMP and stored in sealed 15 mL
falcon tubes to avoid moisture at 4 °C and 37 °C, respectively for total
six months. Stored strips were re-tested at every one month interval for
their reactivity with reference serum samples.

\textbf{Results}

\textbf{Production of rHyC antigen}

The expression of rHyC protein was induced using 1 mM IPTG and culture
was allowed to grow at 37 °C with shaking at 200 rpm for 6 hr. Protein
was purified under denaturing condition using 8 M urea in phosphate
buffer. The homogeneity of purified fusion protein with molecular weight
of about 45 kDa was analysed in SDS-PAGE (\textbf{Fig. 1} ). The
estimated concentration of purified rHyC protein was 94 \selectlanguage{greek}µ\selectlanguage{english}g/mL of elutes
after dialysis.

\textbf{Optimization of Dot-ELISA}

Upon optimization, checkerboard titration revealed the optimal antigen
concentration, serum and conjugate dilution at 188 ng/dot, 1:800 and
1:4000 respectively for clear distinction between positive and negative
serum samples (\textbf{Fig. 2} ). The serum and conjugate dilution were
selected to attain an acceptable well defined dots with less
background/noise signal on strips at the minimum concentration of rHyC
antigen. The antigen volume was selected at 2 \selectlanguage{greek}µ\selectlanguage{english}L to obtain appreciable
size of dot for better visual observation of results.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1} SDS-PAGE analysis showing purified rHyC protein along
with induced culture; Lane 1: 6 h post induction 8M Urea lysate; Lane 2:
purified rHyC; Lane M: Prestained protein marker.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2} Checkerboard titration to standardize rHyC based
dot-ELISA. Antigen concentration (94 ng/1\selectlanguage{greek}µ\selectlanguage{english}L and 188 ng/2 \selectlanguage{greek}µ\selectlanguage{english}L); Serum
(1:400, 1:800 and 1:1200) and conjugate (1:4000 and 1:8000) dilution.

\textbf{Evaluation of Dot-ELISA}

The diagnostic sensitivity and specificity of the assay was assessed
with 109 known positive serum for goat warble fly disease and 92 true
negative serum samples. The developed dot-ELISA provided good parameters
with DSn of 97.3\% and DSp of 95.8\% \textbf{(Table 1).} The rHyC based
dot-ELISA showed strong reactivity with positive serum of GWFI and no
cross-reactivity was observed with positive sera of important parasitic
and bacterial diseases of goats as listed earlier \textbf{(Fig 3).}

\textbf{Table1.} Diagnostic parameters of the optimized rHyC based
dot-ELISA for GWFI.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& & \textbf{Physico-clinical Evaluation} & \textbf{Physico-clinical
Evaluation} & \textbf{Diagnostic Parameters} & \textbf{Diagnostic
Parameters}\tabularnewline
\midrule
\endhead
\textbf{rHyC Dot-ELISA} & + & + & - & \textbf{Total} & \textbf{PPV =
0.96}\tabularnewline
& & 106 & 4 & \textbf{110} & \textbf{NPV = 0.96}\tabularnewline
& - & 3 & 88 & \textbf{91} & \textbf{DSn= 97.3\%}\tabularnewline
\textbf{Total} & \textbf{Total} & \textbf{109} & \textbf{92} &
\textbf{201} & \textbf{DSp= 95.8\%}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3} Specificity and cross reactivity of developed dot-ELISA;
Lane 1: Goat warble fly; Lane 2: Oestrosis; Lane 3: Haemonchosis; Lane
4: Fasciolosis; Lane 5: Amphistomosis; Lane 6: Coenurosis; Lane 7:
Cryptosporidiosis; Lane 8: Paratuberculosis; Lane 9: Enterotoxaemia;
Lane 10: Brucellosis.

A Cohen's kappa index of 0.973 with Standard Error (SE) of 0.019 at very
high significance level (P\textless{}0.001) was observed between the
rHyC based dot-ELISA and microtitre plate indirect-ELISA with a
percentage of agreement of 98.657\% (\textbf{Table 2} ).

\textbf{Table 2.} Evaluation of Cohen's kappa for inter rater
reliability with rHyC based indirect ELISA as reference standard.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& & \textbf{rHyC Dot-ELISA} & \textbf{rHyC Dot-ELISA} &
\textbf{Inter-Rater Relaibility} & \textbf{Inter-Rater
Relaibility}\tabularnewline
\midrule
\endhead
\textbf{rHyC iELISA} & + & + & - & \textbf{Total} & \textbf{P A =
98.657\%}\tabularnewline
& & 68 & 1 & \textbf{69} & \selectlanguage{greek}\textbf{Cohen's κ = 0.973}\selectlanguage{english}\tabularnewline
& - & 1 & 79 & \textbf{80} & \textbf{SE=0.019}\tabularnewline
\textbf{Total} & \textbf{Total} & \textbf{69} & \textbf{80} &
\textbf{149} & \textbf{P\textless{}0.001}\tabularnewline
\bottomrule
\end{longtable}

{[}Strength of agreement: \selectlanguage{greek}κ \selectlanguage{english}index \textless{}0, poor; 0-0.20, slight;
0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, substantial; 0.81-1.0,
almost perfect; SE: Standard Error; PA: Percent Agreement{]}

The assessment of rHyC based dot-ELISA at lower incubation temperature
(18 \textsuperscript{o}C) and time (30 min) provided valid results
suitable for field diagnosis of GWFI with dilutions below 1:10. The
limited assessment of neat and lower dilutions of samples at lower
temperature and incubation period were found to exhibit faint dots at
neat and 1:2 dilution whereas clear distinction was observed at 1:10
dilution of known samples. Moreover, the positive control taken as a
weak positive sample based on microtitre ELISA (OD=0.537) provided valid
results at the above conditions.

Further for serosurveillance, the serum samples available in the ICAR-NF
laboratory were tested, which could clearly differentiate between
infested and uninfested animals. A total of 274 serum samples suspected
of GWFI were tested, out of which 83 samples were found to be positive
for GWFI.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4.} Comparative evaluation with samples of blood (A1-3) and
plasma(B1-3) with weak positive serum(C4-6) as positive control and
negative controls for blood, plasma and serum (D 4-6) under incubation
at 18 \selectlanguage{ngerman}°C for 30 min.

\textbf{Stability of Dot-ELISA strips}

The stability of the dotted strip was assessed with a panel of three
positive and three negative control sera after storage at 4 °C and 37 °C
at 2, 4, 6 months of time period. The results showed clear
differentiation of the dots at both 4 °C and 37 °C storage temperatures
for up to 6 months without losing their reactivity to positive serum
samples \textbf{(Fig 5).}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5} Stability of dotted NCM strips after six months of
storage at 4 \selectlanguage{ngerman}°C and 37 °C. Panel A: Storage of strips at 4 °C; Lane 1-3:
GWFI positive serum; Lane 4-6: Goat negative serum. Panel B: Storage of
strips at 37 °C; Lane 1-3: GWFI positive serum; Lane 4-6: Goat negative
serum.

\textbf{Discussion}

Goat warble fly infestation is an economically important disease of
goats. In India, the disease is prevalent in Union Territory of Jammu
and Kashmir (North India)\textsuperscript{2} and causes huge economic
losses in the region.\textsuperscript{3} Globally, GWFI causes huge
production losses to goat farming in the Mediterranean and Indian
subcontinent with consistent reports from south Italy, Greece, Egypt,
Jordan, Saudi Arabia, Iraq, Iran, Pakistan with variable prevalence
rates over the years.\textsuperscript{4} The development of a sensitive
and specific technique to diagnose warble fly infestation is an
important step towards promoting sustainable control of the
disease.\textsuperscript{30,31}

The diagnosis of GWFI is commonly based on the physicoclinical
observation of subcutaneous warbles at the late stage of L2 and L3 which
occur after 3-4 months of infestation. For early and confirmatory
diagnosis, serological test has been developed for the detection
of\emph{Hypoderma} spp. specific antibodies. Many workers have developed
crude and native HyC based indirect ELISA derived from \emph{H.
lineatum} with DSn 100\%, DSp 92\%;\textsuperscript{21} DSn 98.2\% and
DSp 98.2\%,\textsuperscript{20} respectively. However, it is well known
that crude or native HyC antigen preparation is challenging to
standardize and availability of L1 larvae throughout the year is not
feasible because of the seasonal pattern of the disease and variable
prevalence in a geographical region. An alternative approach is the use
of recombinant protein produced in heterologous system instead of native
antigen. This provides the advantage of purified specific antigen and
amenability to standardization of immunoassay with less background
signal. Recombinant protein based immunoassays overcomes the limitations
associated with available serodiagnostic tests. HyC is the major
immunodominant protein that is present in all members
of\emph{Hypoderminae} . Indirect ELISA based on the rHyC of \emph{H.
lineatum} \textsuperscript{22,20} and \emph{H. bovis}
\textsuperscript{23}have been developed and used successfully for
detecting hypoderma-specific antibodies from cattle serum.

A competitive ELISA based on crude lysate protein derived
from\emph{Przhevalskiana} spp. has been developed for the early
detection of GWFI in goats (8). Microtitre plate ELISA has also been
developed based on \emph{P. silenus} derived recombinant
HyC.\textsuperscript{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. Upon testing the dot-ELISA within a limited trial at ambient
temperature (18 °C) the results were found to be valid and supported the
suitability of the test under field conditions for the diagnosis of GWFI
under sub-tropical and temperate zones. Also, the time required to
perform the present dot-ELISA after blocking of strips is about 1 h 15
min which further supports the field applicability. Several other
studies on parasitic diagnosis based on dot-ELISA require a time period
ranging between 1h 35 min to 3 h after blocking step in various assays
for other parasitic diseases.\textsuperscript{25,26,32} The shorter
incubation time and temperature conditions makes the assay amenable to
field application.

As described earlier, diversity of antigenic preparations have been used
for parasite immunodiagnosis with dot-ELISA such as excretory-secretory
antigens, crude lysate preparations, purified native
antigens.\textsuperscript{25,26} However, in the recent decades,
recombinantly expressed protein antigens have been emphasized through
various studies for their immunodiagnostic
applications.\textsuperscript{24,27,28,32}

In the present study, dot-ELISA was standardized based on rHyC derived
from \emph{P .silenus} for serodiagnosis of GWFI. The expression of rHyC
fusion protein derived from \emph{P. silenus} in prokaryotic expression
system has been previously optimized.\textsuperscript{18} The rHyC
fusion protein was produced in bulk and purified under denaturing
condition and predicted size of purified fusion protein
\textasciitilde{}45 kDa was obtained. The yield of expressed protein
after dialysis was about 3 mg/L of induced culture which is sufficient
to screen approximately 5000 serum samples in triplicate dots.

Moreover, recombinant antigen source provides a sustainable and
consistent source of antigen for diagnostic application all-round the
year thus eliminating the need of larval origin antigens, apart from
providing a diagnostic test with improved parameters. The purified rHyC
protein reacted very well with GWFI positive serum in dot-ELISA format
at an antigen concentration of 188 ng/dot and antibody and conjugate
dilution 1:800 and 1:4000 respectively. The diagnostic sensitivity and
diagnostic specificity of rHyC dot-ELISA were found to be 97.3 \% and
95.8 \%, respectively. The sensitivity and specificity of the developed
dot-ELISA were found comparable as to those of rHyC based
ELISA.\textsuperscript{20,22,23} Recombinant antigen used as purified
antigen helps in reduction of non-specific
reaction.\textsuperscript{33}The assessment at lower time and
temperature conditions for incubation showed variable intensity of dots.
This variation in dot color intensity might be attributed to the prozone
phenomenon as neat samples might present disproportionately high amount
of antibodies in the GWFI positive samples.\textsuperscript{34} Samples
with weaker signals were taken as positive and the dot ELISA was applied
for qualitative assessment of 274 samples for GWFI. The dot-ELISA
optimized in the present study provides a high serum dilution rate at
1:800 which permits the scope of using pooled serum samples for the
assessment of herd screening for warble fly
infestation.\textsuperscript{31,35} The present dot ELISA provides a
high sample dilution rate which improves specificity and may be used
with dried blood sample on filter paper availed in field conditions with
minimal resources for GWFI diagnosis (36). This provides an opportunity
for a standardized simple diagnostic tool for mass surveillance of GWFI
at the national eradication program.

The optimized dot-ELISA was found to be highly specific with GWFI serum
and did not exhibit any visible reactivity with other important
parasitic and bacterial diseases of goats. The rHyC based dot-ELISA was
found to be non-reactive to \emph{Oestrus ovis} positive sera of goats
which is in accordance with the previous observation in western blotting
and microtitre rHyC indirect-ELISA.\textsuperscript{14,18} The optimized
dot-ELISA can serve as economical, efficient, field applicable and thus
forms a suitable tool for serosurveillance of GWFI during disease
control program.

Moreover, blotting technique including dot-ELISA has multiple advantages
over indirect ELISA since protein adsorption is higher in Nitrocellulose
membranes,\textsuperscript{37, 38} which permits the higher dilution of
samples as observed in the optimized assay. The technique provides
simple execution and interpretation with minimal reagents. Also, it can
be used for large volume tests or small number of
samples.\textsuperscript{39, 40}

The cross-reactivity of HyC due to its conserved nature and shared
epitopes among Hypoderminae members has been evaluated in several
studies.\textsuperscript{11, 13, 14, 16, 41, 42} This suggests that the
rHyC based dot-ELISA can be used for serodiagnosis of hypodermosis in
cattle, yalk and cervids with other hypoderminae infestation such
as\emph{H. sinense, H. bovis, H. lineatum, H. diana} and \emph{H.
actaeon} . Antibody kinetics studies for hypoderminae infestation show
that antibodies are observed as early as fourth week of L1 infestation
and persists up to 16 to 20 weeks.\textsuperscript{11} This provides an
opportunity for early diagnosis based on antibody detection and
intervention at L1 stage with prophylactic treatment to avoid production
losses to milk, meat and hide.\textsuperscript{11}

The antigen dotted membranes can be stored for up to 6 months at both 4
°C and 37 °C without any changes in
reactivity.\textsuperscript{27,36,43} This stability of NCM coated
antigen dots makes it more suitable for field application with minimal
storage specification at regional veterinary centers. Further
thermostability studies of antigen dotted NCM strips would be required
for longer time period of storage.

\textbf{Conclusion}

Dot-ELISA has several advantages over commercially available ELISA kits,
including simplicity of the procedure, no requirement of highly precise
equipment such as an ELISA reader, ability to interpret results
visually, low cost and high sensitivity and specificity. The test is
also desired to be amenable to sample pooling to cover herd level
screening through bulk samples which facilitates mass surveillance. The
results of the present study demonstrate that dot-ELISA may provide a
suitable alternative to plate ELISA for the serodiagnosis of GWFI at
regional centres. The present study reports the first dot-ELISA based on
rHyC of \emph{P. silenus} which can support mass surveillance programs
at the field level to achieve sustainable control of GWFI.

\textbf{Acknowledgements}

The authors are grateful to the Indian Council of Agricultural Research
(ICAR), New Delhi, Government of India, for providing ICAR-National
Fellow Scheme to A.Y. The authors are thankful to Sher-e-Kashmir
University of Agricultural Sciences and Technology of Jammu (SKUAST-J)
for providing the facilities. The authors also acknowledge the
ICAR-Central Institute for Research on Goats, Mathura (India) for
providing selected serum samples of goats. The authors are grateful to
the farmers and Veterinary Officers of Union Territory of Jammu and
Kashmir, India.

\textbf{REFERENCES:}

\begin{enumerate}
\tightlist
\item
  Zumpt, F. Morphology, biology and pathogenesis of myiasis-producing
  flies in systematic order. In~\emph{Myiasis in Man and Animals of the
  Old World: A Textbook for Physicians, Veterinarians and Zoologists}
  ~(ed. Zumpt, F.) 205--214 (Butterworths, 1965).
\item
  Yadav A, Katoch R, Khajuria JK, Katoch M, Agrawal R. Prevalence and
  biology of goat warble fly infestation by \emph{Przhevalskiana
  silenus} in Jammu province, India. \emph{Trop Anim Health Prod} .
  2011;43(8):1487--1492.
\item
  Yadav A, Katoch R, Khajuria JK, Katoch M, Rastogi A. Economic impact
  of \emph{Przhevalskiana silenus} infestation in native goats of
  Northern India. \emph{Trop Anim Health Prod} . 2012;44(3):581--587.
\item
  Yadav A, Panadero R, Katoch R, Godara R, Cabanelas E. Myiasis of
  domestic and wild ruminants caused by Hypodermatinae in the
  Mediterranean and Indian subcontinent. \emph{Vet Parasit} .
  2017;243:208--218.
\item
  Otranto D, Puccini V. Further evidence on the internal life cycle
  of\emph{Przhevalskiana silenus} (Diptera, Oestridae). \emph{Vet
  Parasit} . 2000;88(3--4):321--328.
\item
  Liaquat S, Qayyum M, Ahmed H, Arfeen RZU, Celik F, Simsek S.
  Seroepizootiological investigation on Goat Warble Fly Infestation
  (\emph{Przhevalskiana silenus} ) in Pothwar Plateau,
  Pakistan.\emph{Trop Biomed} . 2021;38(2):1-8.
\item
  Oryan A, Razavi SM, Bahrami S. Occurrence and biology of goat warble
  fly infestation by \emph{Przhevalskiana silenus} (Diptera, Oestridae)
  in Iran. \emph{Vet Parasitol} . 2009;166(1--2):178--181.
\item
  Bagheri A, Madani R, Navidpour Sh, Hoghoooghi-Rad N. Prevalence and
  Early Detetion of Hypodermosis in Goats using a Competitive ELISA
  System in Lorestan Province. \emph{Arch Razi Inst} . 2020;Available
  from: https://doi.org/10.22092/ari.2019.125071.1296
\item
  Sayin, F. Incidence and seasonal activity of~\emph{Przhevalskiana
  silenus} ~(Brauer) in angora goats in Turkey. III Symposium on Medical
  and Veterinary Acaroentomology, Gdansk 25--28 September
  1975.~\selectlanguage{polish}\emph{Wiadomości Parazytol.}\selectlanguage{english} ~\textbf{23} , 1--3 (1977).
\item
  Gaglio G, Brianti E, Ferrara MC, Falsone L, Napoli E, Giannetto S.
  Seasonality of goat warble fly infection by \emph{Przhevalskiana
  silenus} (Diptera, Oestridae) in Italy. \emph{Small Rum Res} .
  2016;135:46--49.
\item
  Otranto D, Giangaspero A, Caringella MP, Puccini V. Hypoderma lineatum
  antigen and anti-\emph{Przhevalskiana silenus} antibodies:
  cross-reactivity and antibody kinetics in naturally infested
  goats.\emph{Parassitologia} . 1998;40(3):325--331.
\item
  Santagada G, Otranto D, Traversa D, Faliero S, Puccini V. Comparative
  study of anti-Hypoderma antibody kinetics in sera, single and bulk
  milk samples of naturally infested cattle. \emph{Parassitologia} .
  2001;43(3):109--111.
\item
  Pruett JH, Scholl PJ, Temeyer KB. Shared epitopes between the soluble
  proteins of \emph{Hypoderma} \emph{lineatum} and \emph{Hypoderma
  bovis} first instars. \emph{J Parasitol} . 1990;76(6):881.
\item
  Boulard C, Villejoubert C, Moir\selectlanguage{ngerman}é N. Cross-reactive, stage-specific
  antigens in the Oestridae family. \emph{Vet
  Res.}1996;27(4--5):535--544.
\item
  Otranto D, Boulard C, Giangaspero A, Caringella MP, Rimmele D, Puccini
  V. Serodiagnosis of goat warble fly infestation by\emph{Przhevalskiana
  silenus} with a commercial ELISA kit. \emph{Vet Rec} .
  1999;144(26):726--729.
\item
  Guan G, Luo J, Ma M, et al. Sero-epidemiological surveillance of
  hypodermosis in yaks and cattle in north China by ELISA. \emph{Vet
  Parasitol} . 2005;129(1--2):133--137.
\item
  Asbakk K, Oksanen A, Nieminen M, Haugerud RE, Nilssen AC. Dynamics of
  antibodies against hypodermin C in reindeer infested with the reindeer
  warble fly, Hypoderma tarandi. \emph{Vet
  Parasitol.}2005;129(3-4):323-32.
\item
  Yadav A, Rafiqi SI, Yadav V, et al. First report
  of\emph{Przhevalskiana silenus} derived recombinant hypodermin C based
  indirect ELISA for serodiagnosis of goat warble fly myiasis. \emph{Sci
  Rep.} 2022;12(1):13440.
\item
  Panadero R, López C, Carballo D, et al. Assessment of a recombinant
  antigen versus natural hypodermin C for the serodiagnosis of
  hypodermosis in cattle. \emph{Parasitol Res} . 2000;86(1):67--68.
\item
  Panadero R, López C, Mezo M, Morrondo P, Díez-Baños P. Effect of early
  treatment with ivermectin and doramectin on the dynamics of antibody
  response in cattle naturally infested by \emph{Hypoderma lineatum}
  and\emph{H. bovis} . \emph{Vet Parasitol} . 1997;73(3--4):325--334.
\item
  Webster KA, Giles M, Dawson C. A competitive ELISA for the
  serodiagnosis of hypodermosis. \emph{Vet Parasitol} .
  1997;68(1--2):155--164.
\item
  Casais R, Mart\selectlanguage{polish}ı\selectlanguage{english}n Alonso JM, Boga JA, Parra F. \emph{Hypoderma
  lineatum} : expression of enzymatically active hypodermin C
  in\emph{Escherichia coli} and its use for the immunodiagnosis of
  hypodermosis. \emph{Exp Parasitol} . 1998;90(1):14--19.
\item
  Atelge M, Inci A, Yildirim A, Sozdutmaz I, Adler PH. First molecular
  characterization of hypodermin genes of \emph{Hypoderma bovis} and
  serodiagnosis of bovine hypodermosis with recombinant hypodermin C
  antigen and a synthetic peptide containing its linear B-cell
  epitope.\emph{Vet Parasitol} . 2021;292:109394.
\item
  Varghese A, Raina O, Nagar G, et al. Development of cathepsin-L
  cysteine proteinase based Dot-enzyme-linked immunosorbent assay for
  the diagnosis of \emph{Fasciola gigantica} infection in
  buffaloes.\emph{Vet Parasitol} 2012; 183:382.
\item
  Rold\selectlanguage{ngerman}án W, Cornejo W, Espinoza Y. Evaluation of the dot enzyme-linked
  immunosorbent assay in comparison with standard ELISA for the
  immunodiagnosis of human toxocariasis. \emph{Mem Inst Oswaldo Cruz} .
  2006;101(1):71--74.
\item
  Piña R, Gutiérrez AH, Gilman RH, et al. A dot-ELISA using a partially
  purified cathepsin-L-like protein fraction from \emph{Taenia
  solium}cysticerci, for the diagnosis of human neurocysticercosis.
  \emph{Ann Trop Med Parasitol} . 2011;105(4):311--318.
\item
  Teimouri A, Modarressi MH, Shojaee S, Mohebali M, Rezaian M, Keshavarz
  H. Development, optimization, and validation of an in-house Dot-ELISA
  rapid test based on SAG1 and GRA7 proteins for serological detection
  of~\emph{Toxoplasma gondii} ~infections. \emph{Infect Drug
  Resist.}2019;12:2657-2669.
\item
  Mandal M, Banerjee PS, Kumar S, et al. Development and evaluation of
  serodiagnostic assays with recombinant BgSA1 of \emph{Babesia gibsoni}
  . \emph{Vet Parasitol} . 2014;205(3--4):483--489.
\item
  Landis JR, Koch GG. The measurement of observer agreement for
  categorical data. \emph{Biometrics} . 1977;33(1):159-174.
\item
  Boulard C. Durably controlling bovine hypodermosis. \emph{Vet Res} .
  2002;33(5):455--464.
\item
  Boulard C, Alvinerie M, Argenté G, Languille J, Paget L, Petit E. A
  successful, sustainable and low cost control-programme for bovine
  hypodermosis in France. \emph{Vet Parasitol} . 2008;158(1--2):1--10.
\item
  Zheng W, Zhang R, Wu X, et al. Evaluating troponin C
  from\emph{Psoroptes cuniculi} as a diagnostic antigen for a dot-ELISA
  assay to diagnose mite infestations in rabbits. \emph{Parasit Immunol}
  . 2014; 36:53-59.
\item
  Manoharan S, Parthiban M, Prabhakar TG, et al. Rapid serological
  profiling by an immunocomb-based dot-enzyme-linked immunosorbent test
  for three major poultry diseases. \emph{Vet Res Commun} .
  2004;28(4):339--346.
\item
  Gillet P, Scheirlinck A, Stokx J, et al. Prozone in malaria rapid
  diagnostics tests: how many cases are missed? \emph{Malar J} .
  2011;10:166.
\item
  Boulard C, Villejoubert C. Use of pooled serum or milk samples for the
  epidemiological surveillance of bovine hypodermosis. \emph{Vet
  Parasitol} . 1991;39(1--2):171--183.
\item
  Fisa R, Gállego M, Riera C, et al. Serologic diagnosis of canine
  leishmaniasis by Dot-ELISA. \emph{J VET Diagn Invest} .
  1997;9(1):50--55.
\item
  Costa JG, Vilariño MJ. Semiquantitative dot blot with the GRA8 antigen
  to differentiate the stages of toxoplasmosis infection. \emph{J
  Microbiol Methods} . 2018;149:9--13.
\item
  Gunyakti Kilinc S, Simsek S, Celik F, Kesik HK. Cloning, expression
  and serodiagnostic potential of HSP70 of Taenia multiceps in
  sheep.\emph{Mol Biochem Parasitol} . 2021;245:111397.
\item
  Blanco RM, Takei K, Romero EC. Leptospiral glycolipoprotein as a
  candidate antigen for serodiagnosis of human leptospirosis. \emph{Lett
  App Microbiol} . 2009;49(2):267--273.
\item
  Bosqui LR, Corral MA, Levy D, et al. Evaluation of the Dot-ELISA as a
  diagnostic test for human strongyloidiasis based on the detection of
  IgA in saliva. \emph{Acta Tropica} . 2020;203:105305.
\item
  Domínguez J, Panadero R, de la Fuente-López C, et al. Detection
  of\emph{Hypoderma actaeon} infestation in \emph{Cervus elaphus} with
  ELISA and western blotting. \emph{J Wildlife Dis} .
  2010;46(3):934--938.
\item
  Panadero R, Martínez-Carrasco C, León-Vizcaíno L, et al. Use of a
  crude extract or purified antigen from first-instar cattle
  grubs,\emph{Hypoderma lineatum} , for the detection of anti-Hypoderma
  antibodies in free-ranging cervids from southern Spain. \emph{Med Vet
  Entomol} . 2010;24(4):418-24.
\item
  Hawkes R, Niday E, Gordon J. A dot-immunobinding assay for monoclonal
  and other antibodies. \emph{Anal Biochem} . 1982;119(1):142--147.
\end{enumerate}

\selectlanguage{english}
\FloatBarrier
\end{document}

