Veterinary Parasitology 199 (2014) 24–31

Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

An optimised protocol for molecular identification of Eimeria

from chickens
Saroj Kumar a , Rajat Garg a,∗ , Abdalgader Moftah b , Emily L. Clark c ,
Sarah E. Macdonald c , Abdul S. Chaudhry b , Olivier Sparagano d ,
Partha S. Banerjee a , Krishnendu Kundu a , Fiona M. Tomley c , Damer P. Blake c,∗∗
a
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, Uttar Pradesh, India
b
School of Agriculture, Food and Rural Development, Agriculture Building, Newcastle University, Newcastle upon Tyne NE1 7RU, United
Kingdom
c
Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, North Mymms AL9 7TA, United Kingdom
d
Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Molecular approaches supporting identification of Eimeria parasites infecting chickens
Received 3 July 2013 have been available for more than 20 years, although they have largely failed to replace
Received in revised form
traditional measures such as microscopy and pathology. Limitations of microscopy-led
13 September 2013
diagnostics, including a requirement for specialist parasitological expertise and low sample
Accepted 20 September 2013
throughput, are yet to be outweighed by the difficulties associated with accessing genomic
DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-
Keywords:
Eimeria species identification specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the
Chicken production of a standardised protocol that includes sample collection and DNA template
COCCIMORPH preparation, as well as primer selection from the numerous PCR assays now published. Such
Multiplex PCR a protocol will facilitate development of valuable epidemiological datasets which may be
Nested PCR easily compared between studies and laboratories. The outcome of an optimisation process
Protocol undertaken in laboratories in India and the UK is described here, identifying four steps. First,
samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts
were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a
QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally,
nested PCR was carried out using previously published primers targeting the internal tran-
scribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in
the presence of faecal material, DNA extraction using a traditional phenol/chloroform pro-
tocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using
the morphometric tool COCCIMORPH for the first time with field samples.
© 2013 Dirk Vulpius The Authors. Published by Elsevier B.V. Open access under CC BY-NC-SA license.

1. Introduction

Coccidiosis, caused by protozoan parasites belong-

ing to the genus Eimeria, is one of the commonest and
most economically important enteric diseases of chick-
ens’ worldwide (Shirley et al., 2005). Seven Eimeria species
∗ Corresponding author.
∗∗ Corresponding author. Tel.: +44 1707 666041. can infect the chicken (viz., Eimeria acervulina, Eimeria
E-mail addresses: rajatgarg [email protected] (R. Garg), brunetti, Eimeria maxima, Eimeria mitis, Eimeria neca-
[email protected] (D.P. Blake). trix, Eimeria praecox and Eimeria tenella) and all can

0304-4017 © 2013 Dirk Vulpius The Authors. Published by Elsevier B.V. Open access under CC BY-NC-SA license.
http://dx.doi.org/10.1016/j.vetpar.2013.09.026
 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31 25

compromise economic production and animal welfare, of Eimeria spp. of poultry by morphological analysis
resulting in poor feed conversion ratios, failure to thrive (Castañón et al., 2007).
and elevated mortality (Long et al., 1976; Williams et al., In the present study three different parasite purifi-
2009). Conventionally, identification of Eimeria spp. is cation/DNA extraction procedures (QIAamp Stool Mini
based on morphological features of the sporulated oocyst, kit with and without faecal contamination, and phe-
sporulation time and location/scoring of pathological nol/chloroform) and three different PCR protocols (nested
lesions in the intestine but the procedures involved require PCR ITS-1 amplification and multiplex SCAR PCR in a one or
specialist expertise and have serious limitations due to two tube format) have been tested in India and the UK and
their subjective nature and overlapping characteristics compared to the software tool COCCIMORPH for diagnos-
among different species (Long and Joyner, 1984). Mixed tic efficacy on coccidia positive faecal droppings collected
infections also pose a problem for the precise discrimina- from commercially raised poultry.
tion of species using morphological methods. Alternative
species-specific diagnostics are required to inform routine 2. Materials and methods
animal husbandry, veterinary intervention and epidemio-
logical investigation. 2.1. Faecal sample collection
One such alternative is Eimeria species-specific poly-
merase chain reaction (PCR). Over the last 20 years several During November 2011 to April, 2012, a total of 45 com-
PCR assays have been developed that target genomic mercial poultry farms were sampled from Uttar Pradesh
regions of one or more Eimeria species including the E. and Uttarakhand states of North India. During the same
tenella 5S or small subunit rRNAs (Stucki et al., 1993; period 139 commercial poultry farms in Egypt, Libya and
Tsuji et al., 1999), the first and second internal transcribed the UK were sampled. For collection of poultry droppings
spacer regions (ITS-1 and -2) (Gasser et al., 2001; Lew 50 ml polypropylene conical tubes were used, each with a
et al., 2003; Schnitzler et al., 1998; Su et al., 2003; Woods screw top and containing 5 ml potassium dichromate (2%
et al., 2000) and gene-specific targets including sporozoite w/v). The weight of each tube was recorded and pooled fae-
antigen gene EASZ240/160 (Molloy et al., 1998). In one of cal droppings were collected starting from one corner of a
the most comprehensive studies Fernandez et al. (2003) unit and following a ‘W’ pathway across the unit, collecting
designed species-specific primers for Eimeria spp. from a one fresh dropping every two to five paces depending on
group of SCAR (Sequence-Characterized Amplified Region) the size of the unit until the tube was filled to the 10 ml
markers and used them to develop a multiplex PCR for mark. Three to five tubes were filled per unit. Each tube was
the simultaneous discrimination of different Eimeria spp. then properly capped and the contents were thoroughly
in a single reaction. Importantly, many of these assays have mixed by vigorous shaking. The samples thus collected
been shown to be capable of detecting genomic DNA rep- were transported to the laboratory and refrigerated at 4 ◦ C
resenting as few as 0.4–8 oocyst-equivalents (Fernandez until further processed.
et al., 2003; Haug et al., 2007), or as few as 10–20 oocysts
(Carvalho et al., 2011a; Frölich et al., 2013). Nonetheless, 2.2. Processing of faecal samples
routine application with field samples remains compli-
cated by factors including DNA extraction from within The tubes with faecal material were again weighed and
the tough oocyst wall and faecal PCR inhibition (Raj 1.6 g sodium chloride was added to each tube. Then sat-
et al., 2013). Broader uptake of PCR-based Eimeria diag- urated salt solution was added up to the 25 ml mark. The
nostics can be significantly enhanced by establishment tubes were capped tightly and vigorously shaken until the
of an optimised protocol. Similarly, identification of the faecal material was completely broken and mixed well.
most sensitive and robust primers from the large num- Finally, the tubes were filled up to 50 ml mark with sat-
ber of Eimeria-specific PCR assays that are available is an urated salt solution and mixed thoroughly. On this faecal
essential step towards standardised epidemiological anal- suspension, 1–2 ml of single distilled water was gently
yses appropriate for international comparison. Validation overlaid. The sample was left to stand for ten minutes and
of collection, purification and PCR amplification proto- then centrifuged at ∼750 × g for 8 min. Using a disposable
cols across different labs, in multiple countries, is a key Pasteur pipette, the layer from the interface between the
step in the establishment of optimal sampling strategies saturated salt and the water was transferred to a new 50 ml
as we seek to improve understanding of parasite field polypropylene conical tube. This was continued for three
biology. more times till no material was visible at the interface. The
Beyond PCR other approaches to species-specific new tube was filled up to 50 ml mark with single distilled
identification of Eimeria include quantitative PCR (qPCR) water and centrifuged at ∼750 × g for 8–10 min. The super-
(Morgan et al., 2009; Vrba et al., 2010), although cost natant was carefully removed without disturbing the pellet
is currently limiting for routine applications, and Loop- using a disposable Pasteur pipette, leaving 3–5 ml fluid. The
mediated Isothermal Amplification (LAMP; Barkway et al., supernatant was checked microscopically for unpelleted
2011). Importantly, accessing DNA from within the robust oocysts before discarding.
oocyst wall is a challenge for all of these technologies The sample from the above step was transferred into a
when working with faecal or litter samples. An alternative 2.0 ml microfuge tube, taking care to mix the sample and
computational approach is the use of software tool COC- rinse the sides up to ∼3 cm from the base of the 50 ml tube.
CIMORPH (http://www.coccidia.icb.usp.br/coccimorph), The microfuge tube was then centrifuged at ∼6000 × g for
which is based on identification of sporulated oocysts 5 min and the supernatant was discarded after microscopic
26 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31

screening for unpelleted oocysts. The pelleted oocysts were protocol following disruption using a Mini Beadbeater-8
suspended in 1.0 ml distilled or molecular grade water. as described previously (Blake et al., 2003).
After through mixing, 10 ␮l of this sample was drawn from
the microfuge tube and mixed with saturated salt solu- 2.5. PCR amplification
tion up to the 1 ml mark for estimating the final oocyst
concentration (oocysts per gram of faeces, OPG) in the sam- A summary of the PCR assays tested, and the primers
ple using McMaster chambers. The eimerian oocysts were used, is provided in Supplementary Table 1.
then allowed to sporulate in 2% w/v potassium dichromate
solution at 27 ± 2 ◦ C for three days. Following sporulation, 2.5.1. Identification of Eimeria genus genomic DNA by
the oocysts were thoroughly washed thrice in autoclaved PCR
distilled or molecular grade water for taking photomicro- The presence of Eimeria genus genomic DNA was tested
graphs and pelleted for DNA isolation. by PCR amplification of the partial 18S rDNA sequence
using the primers ERIB1 and ERIB10 as described elsewhere
2.3. Identification of Eimeria spp. by COCCIMORPH (Schwarz et al., 2009). Briefly, each reaction contained
2 ␮l genomic DNA template, 25 pmol forward and reverse
For the identification of eimerian oocysts, photomi- primer, 0.5 U Taq polymerase (Invitrogen, Paisley, UK),
crographs of at least 50 individual sporulated oocysts 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl and 200 ␮M
were randomly taken from each sample at 10×/40× using dNTPs. Standard cycle parameters were 1× (5 min at 94 ◦ C),
a dry high power objective with a photomicrographic 30× (30 s at 94 ◦ C, 30 s at 57 ◦ C, 2 min at 72 ◦ C) and 1×
camera (Moticam5, Hong Kong) attached to a trinocular (10 min at 72 ◦ C). Post-amplification PCR products were
research microscope (Motic Trinocular Research Micro- resolved by agarose gel electrophoresis.
scope BA210, Hong Kong). The identification of Eimeria
spp. of chickens was done using COCCIMORPH soft- 2.5.2. Identification of Eimeria spp. by nested PCR
ware (http://www.coccidia.icb.usp.br/coccimorph/). The The nested PCR protocol using ITS-1 primers was stan-
software was downloaded from the Internet and the oocyst dardised for identification of Eimeria species of poultry.
images (400× magnification) were uploaded for species Primers amplifying the entire ITS-1 sequence with flank-
identification as described online. The Eimeria spp. iden- ing partial 18S rDNA and 5.8S rDNA regions of Eimeria were
tified by the software in each sample was recorded. used in the genus-specific PCR phase, while species-specific
primers targeting the ITS-1 region were used to amplify
2.4. Isolation of genomic DNA
the individual Eimeria species as described elsewhere (Lew
For isolation of genomic DNA, only samples found to et al., 2003).
contain more than 500 (India) or 200 (Egypt, Libya and UK) Briefly, each 25.0 ␮l PCR reaction included 2 ␮l of
OPG were selected for processing. genomic DNA, 25 pmol each of genus-specific primers,
1.25 U of Taq polymerase, 200 ␮M each of dNTPs, and
2.4.1. QIAamp DNA Stool mini kit 2.5 ␮l of PCR buffer containing 1.5 mM MgCl2 . The thermal
Total genomic DNA was isolated using a QIAamp DNA cycling was done with an initial denaturing step at 94 ◦ C
Stool mini kit (Qiagen, Germany) as per the manufacturer’s for 3 min followed by 30 cycles of 94 ◦ C for 30 s, 55 ◦ C for
protocol with some modifications from (i) oocysts purified 30 s and 72 ◦ C for 90 s and a final extension at 72 ◦ C for
as described above or (ii) purified oocysts supplemented 7 min. The product of the primary PCR (1.0 ␮l in 25.0 ␮l
with 100 mg oocyst-negative faecal material collected from reaction mixture) was used as template for the nested
a specific pathogen free chicken to mimic the absence PCR with species-specific primers in individual tubes using
of a flotation step. Briefly, to the pelleted oocysts an the same amplification conditions described above except-
equal volume of autoclaved glass ballotini beads measur- ing different annealing temperatures for different Eimeria
ing ∼0.25–0.5 mm in diameter (Sigma–Aldrich, USA) were spp. (58 ◦ C for E. mitis; 61 ◦ C for E. necatrix and E. praecox;
added and covered with a minimum volume ASL buffer 65 ◦ C for E. tenella; 71 ◦ C for E. acervulina, E. maxima and
(out of total 1.4 ml to be used for DNA isolation) sup- E. brunetti). Negative, no-template controls were included
plied with the DNA extraction kit or sterile TE buffer. The with each assay using triple distilled water in place of tem-
oocysts were then disrupted by vortexing (India; Spinix plate. The amplification of specific nested PCR product was
Vortex Shaker, Tarsons, India; maximum speed) or bead- checked by gel electrophoresis in 2% agarose gels stained
beating (Egypt, Libya and UK, Mini Beadbeater-8, Biospec with 0.5 ␮g/ml ethidium bromide.
Products, Bartlesville, USA; set to homogenise) for two min-
utes. Then, the remaining buffer ASL was added to the tube 2.6. Identification of Eimeria spp. by multiplex PCR
and thoroughly mixed. The suspension was then heated for
5 min at 70 ◦ C and processed as per the QIAamp DNA Stool The multiplex PCR using SCAR primers for identification
kit protocol. The DNA was eluted twice in 100 ␮l TE buffer of the seven Eimeria species that infect chickens (Fernandez
as recommended by the manufacturer and quantified using et al., 2003) was standardised using pure DNA samples from
absorbance at 260 and 280 nm. the Houghton strains of each Eimeria spp.
Initially, the PCR amplification was standardised sepa-
2.4.2. Phenol/chloroform DNA extraction rately for each species using specific primer pairs (0.55 ␮M
Total genomic DNA was isolated from purified for E. tenella, E. maxima and E. mitis; 0.7 ␮M for E. acervulina,
oocysts using a standard phenol/chloroform extraction E. necatrix and E. praecox; 0.85 ␮M for E. brunetti), 200 ␮M
 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31 27

dNTP, 5.0 mM MgCl2 , 3.5 U Taq DNA Polymerase, and 1.6× than 500 OPG were processed, although the number of
amplification buffer (supplied by the manufacturer) in a samples tested at this level was very small.
final volume of 25 ␮l reaction mixture. Thermocycling con-
ditions were set at 96 ◦ C for 5 min for initial denaturation, 3.2. Optimal identification of Eimeria spp.
followed by 30 cycles of 1 min at 94 ◦ C, 2 min at 65 ◦ C
and 90 s at 72 ◦ C, with a final extension at 72 ◦ C for 7 min. Out of 45 poultry farms screened in North India, 37
Once the above conditions were standardised for individ- (82.2%) were positive for Eimeria spp. by microscopic exam-
ual primer pairs, all the primer pairs were put together in a ination with OPG ranging from 0.1 × 103 to 242.5 × 103 . Out
single 50 ␮l reaction mixture for single-tube multiplex PCR of the 37 coccidia positive farms, 30 farms had OPG lev-
with the same cycling conditions as described above. For els above 500 and thus were selected for further Eimeria
two-tube multiplex PCR, amplifications were conducted species identification studies.
separately in two tubes; tube 1 contained the primers for
E. acervulina, E. brunetti and E. mitis while tube 2 contained
3.2.1. COCCIMORPH
primers for E. maxima, E. necatrix, E. praecox and E. tenella.
COCCIMORPH is a computational approach for parasite
All the conditions for PCR remained as described above. The
identification in case of Eimeria spp. from the chicken. Digi-
amplification of specific PCR products were checked by gel
tal images of 50 individual unidentified sporulated oocysts
electrophoresis in 2% agarose gels stained with 0.5 ␮g/ml
of Eimeria spp. were uploaded on to the software. The
ethidium bromide.
software then analysed the oocyst on the basis of dif-
ferent features namely, curvature characterisation, size,
2.7. Statistical analysis symmetry and internal structure characterisation for the
identification of eimerian species. Identification of Eimeria
The results of Eimeria species detection for each assay spp. using COCCIMORPH software revealed the presence
were compared by Chi-square analysis using SPSS version of E. acervulina, E. maxima, E. mitis, E. praecox, E. necatrix
20 (IBM, US). Results were considered significant when and E. tenella, in 96.7%, 36.7%, 90.0%, 3.3%, 23.3% and 16.7%
p < 0.05. of farms, respectively (Fig. 1, Supplementary Table 2). E.
brunetti was not recorded in any of the farms screened
3. Results using COCCIMORPH.

3.1. Genomic DNA extraction 3.2.2. Nested ITS-1 PCR

Nested PCR using ITS-1 primer was standardised with
3.1.1. Protocol selection pure DNA of all seven species of Eimeria. Specific PCR ampli-
Triplicate environmental faecal samples were collected cons of E. acervulina (321 bp), E. brunetti (311 bp), E. maxima
from 30 farms and examined microscopically to confirm US strain (145 bp), E. maxima Australian strain (145 bp),
the presence of Eimeria oocysts (10×/20×). Oocysts were E. mitis1 (328 bp), E. mitis5 (193 bp), E. necatrix (383 bp),
purified, pooled per farm to standardise and split for par- E. praecox (116 bp) and E. tenella (278 bp) were visualised
allel processing by (i) QIAamp DNA Stool kit, (ii) QIAamp (data not shown). In field samples, ITS-1 based nested PCR
DNA Stool kit plus faecal contamination and (iii) phe- identified E. acervulina, E. brunetti, E. maxima, E. mitis, E.
nol/chloroform. Using the Eimeria genus 18S rDNA assay praecox, E. necatrix and E. tenella in 93.3%, 10.0%, 86.7%,
93% (28/30) of the samples processed using the QIAamp 96.7%, 66.7%, 80.0% and 100% farms, respectively (Fig. 1,
DNA Stool kit were PCR positive and 100% of the samples Supplementary Table 2). In 16 farms, both the Australian-
containing ≥5000 OPG at the beginning of the process were and US-type strains of E. maxima were identified, while in
positive (Table 1). The addition of faecal material reduced ten farms only the US-type strain of E. maxima was present.
the PCR positive rate to 30% with only one of 17 samples Similarly, E. mitis was identified by primers specific for both
containing fewer than 20,000 oocysts found to be positive. E. mitis1 and E. mitis5 in all the farms that were positive
Using phenol/chloroform extraction 77% (23/30) samples for E. mitis. Mixed infections of Eimeria spp. were recorded
were PCR positive with a 100% success rate only occurring in all farms with a minimum of at least three species (in
above 20,000 starting OPG. four broiler farms). All seven Eimeria spp. were identified
in three farms.
3.1.2. Protocol sensitivity
The protocol found to be most effective (QIAamp DNA 3.2.3. SCAR multiplex PCR
Stool kit after oocyst flotation) was subsequently tested on Multiplex PCR using SCAR primers was standardised
a larger number of field samples to investigate diagnos- with pure DNA of all seven species of Eimeria. Amplicons
tic sensitivity. In total 139 farms were visited, of which of E. acervulina (811 bp), E. brunetti (626 bp), E. maxima
100 were positive (71.9%) for Eimeria oocysts by micro- (272 bp), E. mitis (460 bp), E. necatrix (200 bp), E. praecox
scopic examination with OPG ranging from 0.2 × 103 to (354 bp) and E. tenella (539 bp) were visualised with indi-
191.3 × 103 . All oocyst positive samples were processed. vidual primer pairs as well as in multiplex PCR (data not
Using the Eimeria genus 18S rDNA assay 96% (96/100) shown). In field samples, the one-tube multiplex PCR could
of the samples were PCR positive and 100% of samples identify E. maxima, E. mitis, E. necatrix, E. praecox and E.
containing ≥5000 OPG were positive (Table 2). Sensitivity tenella, in 16.7%, 3.3%, 43.3%, 3.3% and 13.3% farms, respec-
dropped below 80% only when samples containing fewer tively. E. acervulina and E. brunetti were not identified in any
28 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31

Table 1
Comparison of three DNA extraction protocols for the detection of eimerian genomic DNA within chicken faecal samples by PCR targeting the Eimeria genus
18S rDNA.

OPG n Stool kit +F Stool kit −F Phenol/chloroform −F

<1000 6 0 5 3
1000–5000 5 1 4 3
5001–20,000 6 0 6 4
20,001–100,000 7 3 7 7
100,001–200,000 6 5 6 6
Total 30 9 28 23

OPG, oocysts per gram starting material; Stool kit, QIAamp DNA Stool kit. +F, including contaminating faecal material; −F, without contaminating faecal
material; n, number samples tested per OPG group.

Table 2
The influence of faecal sample oocyst concentration on PCR sensitivity for eimerian genomic DNA. Samples prepared using the optimal oocyst flota-
tion/QIAamp DNA Stool kit DNA extraction protocol with a PCR targeting the Eimeria genus 18S rDNA.

OPG Number farms Theoretical oocysts per PCRa Number positive Percent positive

<500 5 <25 3 60
500–1000 5 25–50 4 80
1001–2000 5 50–100 5 100
2001–5000 10 100–250 9 90
5001–10,000 20 250–500 20 100
10,001–50,000 36 500–2500 36 100
50,001–100,000 15 2500–5000 15 100
100,001–200,000 4 5000–10,000 4 100
Total 100 96

OPG, oocysts per gram starting material.

a
Theoretical oocysts per PCR calculated based upon processing 5 g faeces with DNA elution during extraction in 200 ␮l and inclusion of 2 ␮l per PCR.

Fig. 1. Summary of Eimeria species identification from faecal samples collected on 30 farms in North India. Key as shown in the first panel (Example):
blue = identification by nested ITS-1 PCR, red = COCCIMORPH, yellow = SCAR multiplex (one-tube format), green = SCAR multiplex (two-tube format), neg-
ative (box external to the Venn diagram) = the number of samples not found to contain Eimeria. Data presented in full in Supplementary Table 2. *Denotes
a single E. acervulina result identified by COCCIMORPH and two-tube SCAR multiplex but not nested ITS-1 or one-tube SCAR multiplex as indicated by a
joining broken line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31 29

of the farms screened by one-tube multiplex PCR. A maxi- kit protocol is designed to adsorb substances that can
mum of two Eimeria spp. were identified in six farms, while degrade DNA and inhibit downstream enzymatic reactions
for 11 farms no Eimeria spp. were recorded by one-tube and should minimise PCR inhibition. While it is possible
multiplex PCR. However, two-tube multiplex PCR identi- that the faecal PCR inhibitor concentration over loaded
fied E. acervulina, E. maxima, E. mitis, E. praecox, E. necatrix the InhibitEx matrix it is more likely that the residual
and E. tenella, in 36.7%, 43.3%, 53.3%, 56.7%, 6.7% and 46.7% faecal debris reduced the efficiency of the column purifica-
farms, respectively (Fig. 1, Supplementary Table 2). A max- tion step. In support of this hypothesis comparable studies
imum of five Eimeria species were identified in five farms, using sieved faecal samples with and without flotation
while in two farms no Eimeria spp. were detected by two- were not similarly affected, although this protocol was
tube multiplex PCR. E. brunetti was never identified using not adopted owing to quality control issues avoiding con-
the multiplex PCR in one- or two-tube formats. tamination between samples during processing (data not
shown). Using Eimeria oocysts enriched by flotation in
4. Discussion saturated saline considerably improved PCR sensitivity,
where the Stool kit performed considerably better than
Accurate identification of Eimeria spp. is important not the phenol/chloroform extraction (93% compared to 77%).
only for the diagnosis of disease but also for manage- Extension of these studies to include a larger sample panel
ment of subclinical infection, development and application with the Stool kit revealed an overall sensitivity of 96%,
of effective control strategies, and biological and epi- with 100% accuracy when starting with an OPG in excess
demiological study (Lee et al., 2010; Sun et al., 2009). of 5000 (the equivalent of 250 oocysts per PCR from the
Traditionally, identification of Eimeria spp. has been based beginning of the protocol). DNA precipitation could be con-
on the morphological characteristics of oocysts, parasite sidered to concentrate the DNA template and improve PCR
biology, clinical signs of the affected animals, and the typ- sensitivity, although the additional complexity is likely to
ical macroscopic lesions assessed during necropsy (Long be limiting in a medium throughput surveillance system.
and Joyner, 1984). However, in a natural setting mixed Thus, the low false negative rate and the improved health
infections of different Eimeria spp. are commonly encoun- and safety associated with a non-phenol based protocol
tered and morphological characteristics and pathological supported adoption of the parasite flotation/QIAamp DNA
changes may overlap, hindering accurate diagnosis and Stool kit protocol.
undermining detection of subclinical disease (Long and A comparison of the two most widely studied PCR assays
Joyner, 1984; Rice and Reid, 1973). Thus, it has been sug- for identifying the Eimeria spp. of poultry in field samples
gested that these methods should not be used in isolation (viz., multiplex PCR based on SCAR markers and nested PCR
for differentiation of Eimeria species (Long and Joyner, based on amplification of ITS-1 region of the parasite) was
1984; Lopez et al., 2007). Alternatives include molecular or also made in the present study. Multiplex PCR based on
computational approaches such as PCR, qPCR and the soft- SCAR amplification for the simultaneous identification of
ware COCCIMORPH. PCR assays capable of identifying and Eimeria spp. of the chicken was first described 10 years
differentiating Eimeria spp. have been available for more ago (Fernandez et al., 2003). While the assay performed
than 20 years but, despite recognition as the ‘gold standard’ well with purified genomic DNA its sensitivity and breadth
of detection for many pathogens, this technology is yet of species identification was reduced when applied to the
to replace traditional coccidial diagnostics (Brook et al., field samples in common with previous reports (Frölich
2008; Olano and Walker, 2011; Stucki et al., 1993). Features et al., 2013). Diagnostic multiplex PCR systems used for pri-
of eimerian biology including the resistance of the oocyst mary detection of infectious agents are difficult to optimise
wall to anything other than mechanical disruption, limiting and suffer from inherent disadvantages of low sensitivity
access to template DNA (for most avian-infecting species), and reproducibility, hindering comparison between labo-
and PCR inhibition by the surrounding faecal material have ratories. Additionally, the performance of multiplex PCR
discouraged use of PCR. While several PCR assays have been is directly dependent upon the final concentration of PCR
described to identify specific Eimeria species very few stud- inhibitors and the concentration of DNA of individual infec-
ies have focused on the applicability of these techniques tious agents in the DNA template (Haug et al., 2007). Better
for identifying Eimeria spp. in commercially raised poul- results achieved when dividing the multiplex into two
try throughout the world (Carvalho et al., 2011a,b; Frölich tubes in the present study is notable, offering a compro-
et al., 2013; Haug et al., 2008). Development of a standard- mise between sensitivity and utility in agreement with
ised protocol supporting medium throughput diagnostic Carvalho et al. (2011a). Chi-square analysis of the results
sampling for Eimeria will enhance the value of such data obtained from the field samples using each technique iden-
while promoting the application of PCR and comparison tified significant differences between all assays (p < 0.05),
between studies. illustrating the importance of selecting and retaining a sin-
Following collection of fresh environmental faecal sam- gle, standardised procedure if comparable results are to
ples we explored two DNA extraction procedures and the be generated. Application of the ITS-1 nested PCR assay
influence of residual faecal contamination. The inclusion of described previously by Lew et al. (2003) identified more
faecal material dramatically reduced PCR sensitivity with Eimeria spp. from more farms, benefiting from a multi-copy
genomic DNA purified using the QIAamp DNA Stool kit, genomic target and a nested PCR strategy. The require-
supporting the value of even a rudimentary pre-extraction ment for two PCR steps adds complexity, time and expense
parasite purification step. The cause of this inhibition to the nested assay but the improved sensitivity was
remains unclear at present. The InhibitEx step of the Stool distinct.
30 S. Kumar et al. / Veterinary Parasitology 199 (2014) 24–31

Molecular identification of Eimeria spp. using PCR was easy to interpret output can improve uptake in developed
supplemented during these studies by the online COC- and developing regions. As the cost of PCR equipment and
CIMORPH tool, an innovative approach developed for reagents continues to drop, it is feasible that the proto-
identification of eimerian oocysts of poultry and rabbits in cols described here will be developed and integrated into
which digital images of unidentified sporulated eimerian routine poultry management and veterinary surveillance.
oocysts are uploaded for species identification on the basis
of sporulated oocyst morphology (Castañón et al., 2007). Acknowledgements
COCCIMORPH was most effective with E. acervulina and E.
mitis, demonstrating good agreement with the nested ITS- Authors are thankful to the Indian Council of Agri-
1 PCR assay, although it fared less well with E. brunetti, E. cultural Research, New Delhi and the Director, Indian
praecox and E. tenella. Indeed E. brunetti was not identi- Veterinary Research Institute, Izatnagar for providing nec-
fied in any sample, although the occurrence of this species essary facilities. The financial assistance provided by DFID
was found to be low throughout the study. Perusal of avail- and BBSRC, UK in the form of CIDLID project BB/H009337
able literature revealed that no data exists on the use of (Anticoccidial vaccine development: the importance of
this software for identification of Eimeria spp. in field sam- genetic diversity and delivery strategy) and the Libyan Gov-
ples. It has long been recognised that the size and shape ernment for the PhD studentship awarded to A. Moftah is
ranges of eimerian oocysts are wide, overlap substantially duly acknowledged. This manuscript has been assigned the
between species (Long et al., 1976) and may vary due to reference PPB 00587 by the RVC.
environmental and physical factors (Jones, 1932; Joyner,
1982). Further, infrequent species can remain undetected
using COCCIMORPH given that a small subsample may not Appendix A. Supplementary data
present a true representation of the total sample. As such,
while COCCIMORPH can be a valuable tool for prelimi- Supplementary data associated with this article can be
nary screening/identification purposes or in the absence found, in the online version, at http://dx.doi.org/10.1016/
of a laboratory it should be reinforced with microscopic or j.vetpar.2013.09.026.
molecular validation.
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