Biotechnology Advances 24 (2006) 590 – 603

www.elsevier.com/locate/biotechadv

Biotechnological advances in the diagnosis of avian coccidiosis

and the analysis of genetic variation in Eimeria
G.M. Morris, R.B. Gasser ⁎
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia
Received 17 April 2005; received in revised form 18 June 2006; accepted 18 June 2006
Available online 28 June 2006

Abstract

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This
disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the
different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major
problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated
with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been
significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews
the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction
(PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of
Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of
this important disease of poultry.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Coccidiosis of chickens; Eimeria; Prevention; Vaccination; Diagnosis; Control

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
2. Biology, life-cycle and pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
3. Distribution, and control through chemotherapy and vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
4. Advances in the diagnosis of coccidiosis and analysis of genetic variation in Eimeria. . . . . . . . . . . . . . . . 593
4.1. Multilocus enzyme electrophoresis (MEE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
4.2. Southern blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
4.3. Pulsed-field gel electrophoresis (PFGE) and related methods for the analysis of chromosomal DNA. . . . . 595
4.4. Polymerase chain reaction (PCR)-coupled methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
4.4.1. ‘Fingerprinting’ techniques for the analysis of genetic variation and definition of specific genetic markers 596
4.4.2. PCR-based detection methods using genetic markers in nuclear ribosomal DNA . . . . . . . . . . 598

⁎ Corresponding author. Tel.: +61 3 97312000; fax: +6 13 97312366.

E-mail address: robinbg@unimelb.edu.au (R.B. Gasser).

0734-9750/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.06.001
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603 591

4.4.3. PCR-coupled manual and semi-automated electrophoretic methods for the diagnosis and/or analysis
of genetic variation within and among species of Eimeria . . . . . . . . . . . . . . . . . . . . . 599
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601

1. Introduction prospects that these methods provide toward a better

Coccidiosis of chickens is an enteric parasitic disease
caused by multiple species of the protozoan parasite genus
Eimeria (Apicomplexa: Eucoccidia: Eimeriidae) and is
one of the commonest and economically most important
diseases of poultry world-wide (Shirley et al., 2005). It
causes production losses, and high morbidity (due to
acute, bloody enteritis) and mortality rates. While the
control of coccidiosis has relied mainly on the preventa-
tive use of anticoccidial drugs (coccidiostats), together
with the induction of species-specific natural immunity in
chicken flocks (Shirley et al., 2004, 2005), this widely
used approach is costly and has led to serious problems
with drug resistance in Eimeria populations (Williams,
1998). Due to these drug resistance problems, live, viru-
lent vaccines have been utilized to protect chicken flocks
against coccidiosis, particularly in intensive establish-
ments (Williams, 2002b), and attenuated or precocious
live vaccines are now finding wide application (Shirley
et al., 2004, 2005). In addition to the use of preventative
chemotherapy and vaccination, the specific diagnosis of
infection plays a key role in the prevention, surveillance
and control of coccidiosis. Traditionally, diagnosis has
been achieved by detecting and/or enumerating Eimeria
oocysts excreted in the faeces from infected chickens and
measuring oocyst/sporocyst dimensions, and (at post-
mortem) assessing the site and extent of the pathological
lesions caused by Eimeria in the intestine of chickens
(Long and Joyner, 1984). However, these approaches can
be unreliable, particularly given that multiple species of
Eimeria can simultaneously infect the host and because
there can be an “overlap” in the sizes of oocysts and the
sites of infection in the intestines for some species (Long
and Joyner, 1984). Increasingly, molecular tools have
been developed and relied upon for the diagnosis of
coccidiosis. The purpose of this article is to provide a brief
background on the biology and life-cycle of Eimeria
species of chickens and the disease they cause, to briefly
describe methods of control, including vaccination, and to
review traditional and molecular methods for the
diagnosis of coccidiosis in chickens and the analysis of
genetic variation within and among species of Eimeria,
considering their relative merits, and emphasizing the
understanding and control of coccidiosis.
2. Biology, life-cycle and pathogenesis
Seven species of Eimeria (E. acervulina, E. brunetti, E.
maxima, E. mitis, E. necatrix, E. praecox and E. tenella)
are recognized, causative agents of coccidiosis in chickens,
and these species differ in their pathogenicity (Williams,
1998; McDougald, 2003; Shirley et al., 2005). The life-
cycles of all of these species are homoxenous. Chickens
ingest sporulated oocysts from contaminated litter, and
these pass into the intestinal tract, where sporozoites are
released and subsequently invade the cells of the intestinal
wall (McDougald, 2003). Here, the developing schizonts
undergo at least two generations of asexual reproduction;
these replicative phases result in cellular damage in the
epithelium, particularly the later schizont stages which
form in the lamina propria. After at least two generations of
asexual reproduction, gametes are formed, their fusion
then producing a zygote. This matures into an oocyst and is
subsequently excreted in the faeces. Given the correct
environmental conditions (warmth, oxygen and moisture),
the oocyst sporulates and becomes infective (Williams,
1998; Allen and Fetterer, 2002). The entire cycle usually
takes 4–6 days, depending on the species. This short,
homoxenous life-cycle, combined with the potential for
massive reproductive capability during the intracellular
phase, makes this group of parasites a serious problem
under intensive farming conditions.
It is the asexual replicative phases that lead to most
damage to the intestinal tissues, causing varying degrees
of digestive disturbances, fluid and blood loss, and sus-
ceptibility to other diseases (McDougald, 2003). Indi-
vidual birds may show no clinical signs, suffer a mild
loss of appetite, decreased weight gain or weight loss,
diarrhoea (which can be bloody), dehydration and death.
Immunity develops rapidly and infections can be self-
limiting, but naive birds which consume large numbers
of oocysts can be severely affected and die. Immunity is
strictly species-specific, and birds exposed to one species
remain susceptible to infections with heterologous species
of Eimeria, although there can be limited cross-protection
between some strains of a species (e.g., E. maxima) (see
592 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
Shirley et al., 2005). The pathogenicity of the seven
species of Eimeria varies, with some developing deep in
the intestinal mucosa, causing wide-spread damage and
distinct gross lesions (e.g., E. tenella) and others being
less destructive but still having a significant impact on
production. For example, E. mitis and E. praecox are
sometimes erroneously considered to be virtually innoc-
uous. However, E. praecox is known to reduce weight
gain and feed conversion efficiency, even though no
morbidity may be observed (Gore and Long, 1982;
Williams, 1998). Infection with E. mitis usually produces
indistinct lesions, but may still result in reduced weight
gain and morbidity (Fitz-Coy and Edgar, 1992; Williams,
1998). Therefore, all species are potentially of economic
importance.
3. Distribution, and control through chemotherapy
and vaccination
The seven species of Eimeria in chickens are present
world-wide and are of paramount importance in intensive
farming situations (Williams, 1998). Williams (1999)
reported that it was exceedingly rare to find any com-
mercial chicken flock not affected by Eimeria. For exam-
ple, a survey of broiler farms in Brazil and Argentina
found viable oocysts in 89 of the 90 farms examined
(McDougald et al., 1987). A subsequent survey of broiler
farms in North and South America found that almost all of
them had Eimeria present (McDougald, 2003). In addition
to the genus being ubiquitous, multiple species frequently
affect a single poultry operation. Surveys have shown that
infection with two or more species is typical (e.g.,
McDougald et al., 1986). Up to six species have been
shown to occur simultaneously on a single farm (Williams
et al., 1996; personal observations). Hence, the ubiquity of
chicken Eimeria precludes eradication as a practical
option for control on intensive poultry farms. Since
species-specific immunity develops rapidly, the manage-
ment of coccidiosis aims to achieve a balance between
allowing natural immunity to build up and preventing high
oocyst exposure to naïve birds, which would result in overt
disease (Shirley and Bedrnik, 1997). Hygiene, antic-
occidial drugs and vaccines all play key roles in control.
As species of Eimeria have homoxenous life-cycles,
mechanical transmission is the primary means of spread
between farms and between sheds on a farm (McDougald,
2003). Oocysts are resistant in the environment, both to
climatic extremes and disinfectants, surviving for weeks
or months in soil, but they may last for only days in litter
when exposed to heat caused by fermentation and/or
ammonia from faecal material. Williams (1995) found
that after only 5 days, most E. acervulina oocysts seeded
into litter were disintegrating, although viable oocysts
were still detected at 23 days. Good hygiene, such as
cleaning boots and exchanging clothes between sheds,
and the eradication of rodents, assists in minimizing the
transmission of oocysts. Effective farm management,
such as well maintained, drip-free water lines, minimizes
the level of infective oocyst contamination in the litter, as
desiccation significantly reduces sporulation.
Chemotherapy has been the main approach for con-
trolling coccidiosis in chickens (Allen and Fetterer, 2002).
Anticoccidial drugs are usually used preventatively; if a
farmer was to wait for overt signs of disease before treat-
ing the flock, morbidity and mortality would be high and
the economic damage would already be done. Almost all
commercial, intensively farmed chickens are adminis-
tered anticoccidial drugs prophylactically. When given at
accurate preventative (low) doses, Eimeria species are
able to complete their life-cycle without large numbers of
infective oocysts building up in the environment. Such
subclinical infections result in the development of strong,
specific natural immunity without overt disease (Trees,
2002; McDougald, 2003; Shirley et al., 2005).
The efficacy of anticoccidial drugs has been the pri-
mary factor allowing modern commercial farming of
poultry to develop. Without preventative medication regi-
mes, it has been considered to be almost impossible to rear
large numbers of birds under intensive conditions (Chap-
man, 1997). However, there are increasing, serious pro-
blems associated with the wide-spread reliance on
anticoccidial drugs to control (i.e., suppress) coccidiosis.
There are now mounting concerns about drug residues
and raising public awareness about the use of drugs in
food production animals (Williams, 2002a). Importantly,
genetic resistance in Eimeria against anticoccidial drugs
is widespread and has developed to all compounds cur-
rently in use (Chapman, 1997). In addition, such drugs
cannot be used prophylactically after the growing period
of breeders or layers, because of the accumulation of
residues in the eggs produced (Shirley et al., 1995; Shirley
and Bedrnik, 1997). Currently, the only practical alter-
native to the reliance on anticoccidial drugs is the use of
live vaccines to protect chicken flocks against coccidiosis
(Chapman et al., 2002).
Live (sporulated oocyst) vaccines based on virulent
strains/species of Eimeria include Coccivac R D,
CoccivacR E, ImmucoxR ADVENTR and NobilisR
COX-ATM (Shirley et al., 1995, 2005; Shirley and
Bedrnik, 1997; Williams, 1998, 2002a,b). Since the host's
immunity is specific to a particular species of Eimeria,
vaccines must contain strains of each relevant species
(usually 3–7) in order to be effective. When chicks are
vaccinated with a virulent, wild-type strain, the initial
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
application is with a relatively low dose of oocysts, and
should be uniform across the flock. This strategy of
“controlled exposure” allows protective immunity to
develop before the contamination of litter with oocysts
becomes high. However, if there is asynchronous infec-
tion within the flock (due to incorrect vaccine applica-
tion), some naive birds may still ingest large numbers of
oocysts, leading to clinical disease (Shirley and Bedrnik,
1997). In addition, the virulent vaccine strain, while given
in controlled doses to minimize pathogenic effects, still
undergoes the normal replicative cycles in the intestine
and causes some degree of tissue damage. Vaccination
may therefore result in a weight gain setback (Williams,
1998), which is less important for valuable birds with a
long life expectancy, such as layers or broiler breeders, but
is less justifiable economically for short-lived broilers.
More recently, live attenuated (sporulated oocyst)
vaccines containing 3–7 species of Eimeria have been
released. These include LivacoxR Q, LivacoxR T, ParacoxR,
ParacoxR 5, EimeriavaxR 4 m, and EimeriavaxR Plus
(Shirley et al., 1995; Shirley and Bedrnik, 1997; Williams,
1998, 2002a,b) and are also of major biotechnological
importance in many countries. These attenuated strains have
been selected for rapid passage (i.e., having a shortened pre-
patent period) through the chicken host (Shirley and Bedrnik,
1997). Consequently, they have a low reproductive potential
and have a reduced virulence, but still have a strong immu-
nogenicity (Shirley et al., 1995; Shirley and Bedrnik, 1997;
Williams, 1998). These changes are stable and inheritable
(Shirley et al., 1995; Shirley and Bedrnik, 1997; Williams,
1998). For instance, chickens vaccinated with ParacoxR
have been reported to have an improved feed conversion
ratio efficiency and a lower mortality compared with
unvaccinated, drug-treated control chickens. These char-
acteristics demonstrate that live, attenuated/precocious
vaccines are suitable for use in broiler flocks (Chapman et
al., 2002). An added advantage of attenuated, live vaccine
lines is that they should interbreed with wild-type strains
already present in poultry establishments, resulting in local
populations of Eimeria which are less pathogenic and more
drug-sensitive (Williams, 1998; Chapman et al., 2002).
In practice, vaccination regimes can be combined
with anticoccidial drug use to protect birds before
resistance develops, but to still allow Eimeria to replicate
(“cycle”) and stimulate immunity. The synchronous use
of medication also “shields” against species of Eimeria
not included in the vaccine applied (Chapman et al.,
2002). In addition, a combined vaccination/drug treat-
ment regimen may also extend the useful life span of
anticoccidial drugs, by the process of attenuated strains
mixing with field strains (cf. Williams, 1998). While
live, attenuated vaccines provide the best prospects for
593
the control of coccidiosis in the immediate future,
specific, well-defined recombinant (genetically engi-
neered) immunogens may have potential in the longer
term (Chapman et al., 2002; Shirley et al., 2005). The
production of live vaccines via propagation in chickens
is expensive, the vaccines have a limited shelf-life and
they can be readily contaminated during production and/
or processing.
4. Advances in the diagnosis of coccidiosis and
analysis of genetic variation in Eimeria
As emphasized in Section 1, the accurate diagnosis of
coccidosis in a chicken flock is central to its effective
control. This applies to routine monitoring, for the
investigation of a disease outbreak consistent with
coccidiosis and for ensuring the effectiveness of the
application of a live vaccine. In theory, several different
phenotypic traits can be utilized simultaneously for the
identification of, and differentiation among, the seven
species of Eimeria. Each species tends to infect a
specific site in the intestinal tract, usually producing
lesions of characteristic gross appearance. In addition,
the size and shape of oocysts can vary among the species.
For example, E. tenella reproduces in the caeca and
adjacent intestine, and produces bloody lesions. E.
maxima and E. necatrix both develop in the mid-small
intestine, but should in theory be distinguishable based
on the different sizes of their oocysts. Experimentally
“passaging” oocysts through chickens provides relevant
information regarding pre-patent periods and immuno-
genicity against reference strains (Shirley et al., 2005). In
practice, species cannot be identified to species and
distinguished unequivocally based on the above criteria
alone (Long and Joyner, 1984). Multiple species
frequently infect a single host, leading to an overlap of
the affected zones in the intestines and variation in the
nature and extent of lesions. Also, there can be
considerable overlap in oocyst size and shape (Long
and Joyner, 1984). Hence, traditional methods are not
sufficiently reliable to allow a species-specific diagnosis
of coccidiosis to be made. Some of the limitations of the
traditional methods have been overcome by the deve-
lopment of various biochemical and molecular methods
(see Table 1), which are reviewed in the following sections.
4.1. Multilocus enzyme electrophoresis (MEE)
The principle of multilocus enzyme electrophoresis
(MEE) is that non-denatured proteins (e.g., enzymes) may
be separated by their migration through a gel under the
influence of an electric field (reviewed by Andrews and
594 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603

Table 1
Summary of selected biochemical and molecular methods developed and employed for the detection or identification of Eimeria species of chickens,
for the analysis of genetic variation within and among species and for the specific diagnosis of coccidiosis
Method
Multilocus enzyme electrophoresis (MEE)
Southern blot analysis
Pulsed-field gel electrophoresis (PFGE)
Field inversion gel electrophoresis (FIGE)
Amplified fragment length polymorphism
(AFLP)
Random amplification of polymorphic
DNA (RAPD) or arbitrarily-primed PCR
(AP-PCR)
Sequence-characterized amplified
regions (SCARs)
Specific PCR detection using primers
to nuclear DNA
PCR-based electrophoretic methods for
diagnosis and the analysis of genetic
variation, utilizing the first and second
internal transcribed spacers of ribosomal
DNA (ITS-1 and ITS-2, respectively)
Principle and DNA target region Selected articles
Separation of proteins/enzymes by electrophoresis. Shirley (1975),
Andrews and Chilton (1999)
Restriction enzyme-digested genomic DNA separated Southern (1975),
by electrophoresis, transferred to a membrane and then Ellis and Bumstead (1990)
subjected to hybridization using radiolabelled probes.
Electrophoretic separation of large molecules Carle et al. (1986),
(chromosomes) by application of an intermittent Shirley et al. (1990)
electric field.
Electrophoretic separation of large molecules Carle et al. (1986), Fernando
(chromosomes) by application of a periodically and Pasternak (1991)
inverted electric field.
Selective, specific enzymatic amplification following Vos et al. (1995), Shirley and
restriction endonuclease digestion of genomic DNA Harvey (2000), Blake et al. (2003)
and ligation with adapters.
Enzymatic amplification of genomic Welsh and McClelland (1990),
DNA fragments using short oligonucleotide Williams et al. (1990), Shirley
primers of arbitrary sequence. and Bumstead (1994),
MacPherson and Gajadhar (1993)
RAPD-derived bands are isolated, cloned and then Fernandez et al. (2003a,b, 2004)
sequenced. Primers can be designed to the sequence(s)
for their specific PCR-amplification (at high stringency).
Single primer pair targeting the 5S ribosomal RNA Stucki et al. (1993)
(E. tenella).
Single primer pair targeting a sporozoite Molloy et al. (1998)
antigen gene (E. acervulina).
Separate primer pairs targeting the ITS-1 Schnitzler et al. (1998, 1999),
(for each of multiple species of Eimeria). Su et al. (2003), Lew et al. 2003
PCR-coupled restriction fragment length polymorphism Woods et al. (2000b)
analysis (PCR-RFLP): electrophoretic analysis of
restriction endonuclease-digested radiolabelled
ITS-2 amplicons in a denaturing polyacrylamide
gel matrix (for six species of Eimeria).
Denaturing polyacrylamide gel electrophoresis (DPGE) Woods et al. (2000a)
and single-strand conformation polymorphism (SSCP)
analyses for the display sequence variation both within
and among amplicons, produced from the ITS-1
and ITS-2 ribosomal DNA regions
using a single set of genus/family-specific,
radiolabelled oligonucleotide
primers (for five species of Eimeria).
Electrophoresis of amplicons produced from the ITS-2 Gasser et al. (2001, 2005)
using a single primer set (one of which is
fluorescently-labelled). Can be used for the specific
identification of and differentiation among all seven
currently recognized species of Eimeria, and the
detection of variation within species.

Chilton, 1999). Homologous enzymes may differ in
charge and size, due to underlying variation in the
originally transcribed DNA sequence. As the rate of a
protein's migration through a gel under an electric field
also varies with charge and size, enzymes from different
individuals or species may be separated. Their relative
positions can then be revealed by appropriate enzyme-
specific staining (Andrews and Chilton, 1999). The
equipment and specific stains necessary for MEE are in
common use, and this technique has been successfully
applied to investigate genetic variation within and among
species of Eimeria infecting chickens.
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
For instance, Shirley (1975) used MEE to study 11
strains of 6 species of chicken Eimeria (one ‘species’
being E. acervulina var. mivati, now recognized as
being indistinguishable from E. acervulina). Each of the
species examined could be identified and differentiated
using four distinct enzymes, and the author discovered a
degree of intra-specific variation. While this initial study
was conducted on laboratory strains, MEE was also
shown to be effective for the analysis of mixed field
samples. Chapman (1982) examined the electrophoretic
mobility of glucose phosphate isomerase (GPI) in E.
acervulina, E. praecox and ‘E. mivati’ and compared the
electrophoretic banding profiles of passaged Eimeria
taken from poultry farm litter with laboratory reference
strains. These profiles were used to successfully distin-
guish among species/strains, provided each constituted a
minimum of 10–20% of the oocysts in the original sample.
Numerous laboratories have since utilized MEE as a
means to aid in the differentiation among Eimeria species.
This approach has been shown to be effective for samples
taken from around the world (Shirley et al., 1989), in-
cluding Australia (Andrews et al., 1990), Canada and the
U.S.A (Johnston and Fernando, 1997), Japan (Nakamura
et al., 1986), and Sweden (Thebo et al., 1998). In the
detection of different enzymes separated by gel electro-
phoresis, MEE utilizes a well-established system that has
been successful in many other situations. Compared with
other organisms, coccidia are well suited to this technique;
large numbers of oocysts can be collected and pooled
from faeces, and, if needed, they can be passaged through
chickens to amplify oocysts from a small initial sample
(Long and Joyner, 1984). However, there are some sig-
nificant limitations. Large number of oocysts may not
always be available, and passaging adds another expense
and delays the test result (Chapman, 1982; Procunier et
al., 1993; Fernandez et al., 2003b). The process is awk-
ward and time-consuming (Procunier et al., 1993;
Fernandez et al., 2003b). There is strong selection pres-
sure for stability in protein function, resulting in relatively
limited variation in a protein's amino acid sequence; as
MEE detects such variation, its application is restricted
because of the limited magnitude of variation present
(Shirley, 1994a; Fernandez et al., 2003a,b). In addition,
even if there is underlying variation in the DNA sequence,
this may not translate into a variation in amino acids, and
even if it does, this may not result in varying mobilities
during electrophoresis (Shirley, 1994a).
4.2. Southern blot analysis
Southern (1975) developed a method for the detection
of specific fragments of DNA separated by electropho-
595
resis. Genomic DNA is digested using one or more res-
triction endonucleases. The resultant fragments are
separated on a gel by electrophoresis, hybridized to one
or more specific probes, and their positions detected by
autoradiography or fluorography. This method's repro-
ducibility is based on the ability of restriction enzymes to
cleave DNA at specific recognition sites in a nucleotide
sequence. For example, for the nucleotide sequence 5′-
GGATCC-3′, the restriction enzyme BamH1 will invari-
ably cut the DNA strand between the two guanine bases.
Southern blot hybridization (coupled to restriction
fragment length polymorphism analysis, RFLP) was first
applied to Eimeria species of chickens by Ellis and
Bumstead (1990). Genomic DNA of E. tenella, E. acer-
vulina or E. necatrix was digested with the endonuclease
EcoRI and probed with an RNA probe (which was pre-
viously prepared from total cellular RNA extracted from
sporulated oocysts). The three species could be readily
differentiated. Restriction fragment analysis using the
endonuclease BamH1 and a pRibI probe could not distin-
guish among four different strains of E. tenella (Hough-
ton, Weybridge, WIS and Doran) (Ellis and Bumstead,
1990). However, a more recent study did reveal variation
within this species. Shirley (1994a) examined seven
strains of E. tenella using a panel of endonucleases (in-
cluding HinfI, RsaI, HpaII and EcoRI). Four probes were
derived from repetitive sequences of genomic DNA of E.
tenella and used to hybridize to the restriction fragments
produced via separate digestions with the endonucleases.
Variation was detected among strains of E. tenella,
including the Houghton and WIS strains, for which no
variation was previously detected (cf. Ellis and Bumstead,
1990). The detection of such intra-specific variation sug-
gested that Southern blot analysis was more sensitive than
MEE (Shirley, 1994a). While the technique employs
common reagents, an appropriate probe must be designed
to hybridize specifically to the digested DNA fragments.
Other limitations include the complexity (involving more
steps than allozyme electrophoresis), and the need for
relatively large numbers of oocysts to process (Procunier
et al., 1993; Shirley, 1994a; Fernandez et al., 2003b).
4.3. Pulsed-field gel electrophoresis (PFGE) and
related methods for the analysis of chromosomal DNA
Traditional methods of gel electrophoresis have a
limited ability to separate DNA molecules of above a
certain threshold size (greater than 15 to 20 kilobases)
(Carle et al., 1986). These large molecules tend to migrate
at the same rate, regardless of size. However, by making
minor alterations to the electrophoretic protocol, DNA
molecules of 50 kb to several Mb can be separated. One
596 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
method is to use ‘pulsed-field gel electrophoresis’
(PFGE), where an electric current is applied intermittently
across the gel, encouraging migration of large molecules.
The position of the DNA molecules is then detected by
staining with ethidium bromide or by hybridization to a
radioactive probe (Fernando and Pasternak, 1991; Shirley,
1994b).
The chromosomes of Eimeria fall within the
appropriate size for analysis by PFGE, and have been
investigated. For example, Shirley et al. (1990) used this
method to characterize the karyotype of E. tenella.
While it had been suggested that the haploid E. tenella
nuclear genome had 5 chromosomes, PGFE revealed at
least 12. A subsequent study (Shirley, 1994b) also
revealed differences in the karyotype within E. tenella,
uncovering chromosomal size variation between strains.
In addition, particular probes were found to bind to
some individual chromosomes, but not others, revealing
basic sequence differences between homologous pairs.
Field inversion gel electrophoresis (FIGE) is similar
in most respects to PFGE, except that the electric field is
inverted periodically (alternating between ‘forward’ and
‘reverse’) (Carle et al., 1986). A large molecule
migrating through a gel will have a ‘directional’
conformation: it will form a wedge-shape, with the
leading edge being more narrow than the following bulk.
When the direction of the electric field is altered, the
molecule will change conformation, with the wedge-
shape gradually inverting to the opposite end of the
molecule (the new ‘leading edge’), before significant
migration can occur (Carle et al., 1986). The conse-
quence of this is that by periodically switching the
direction of the electric field, and balancing the periods
of ‘forward’ and reverse’ migration, the movement of
large molecules can be made to be strongly size-depen-
dent. Two voltage sources may be connected to the
electrophoretic apparatus to allow the forward and re-
verse fields to be run at different voltages. Fernando and
Pasternak (1991) used this method to study the mole-
cular karyotype of five species of Eimeria from chickens.
Comparing single samples representing each species,
they discovered that each species possessed a distinctive
pattern, which was readily distinguishable from that of
each other species examined. However, they did not
investigate possible variation within each species of
Eimeria.
The various electrophoretic “karyotyping” methods
provide broad information about the genome, such as
chromosome size and minimum number. Additional
information can be obtained from the differential
binding to DNA probes of chromosomes previously
separated by PFGE (Shirley, 1994b). However, PFGE
cannot provide detailed information about the genome
and is very time-consuming to carry out; it can take
more than 10 days for a gel to run (Shirley, 1994b).
4.4. Polymerase chain reaction (PCR)-coupled methods
A limitation of the molecular methods described in
Sections 4.1–4.3 is that relatively large amounts of para-
site material are required for effective analysis and/or
detection. In some cases, this may require the propagation
of the parasite(s) (‘passaging’) in live chickens to produce
sufficient numbers of oocysts, which can be time-consu-
ming, laborious and costly. Clearly, the advent of the
polymerase chain reaction (PCR) (Saiki et al., 1985, 1988)
overcame this limitation and has enabled the analysis of
tiny amounts of parasite material.
The PCR enables the selective amplification of DNA
from complex genomes. In brief, the principle of the
technique is that the double-stranded genomic DNA tem-
plate is denatured by heating, and the temperature is then
decreased to allow one or more oligonucleotide primers to
hybridize (anneal) to their complementary sequences on
opposite strands of the template; the template-directed
DNA synthesis (extension) then proceeds in both orien-
tations from the primer sites by enzymatic catalysis with a
thermostable DNA polymerase and results in the
production of double-stranded products. This synthesis
is usually repeated 20–40 times in an automated thermal-
cycler. In each cycle, the template is replicated by a factor
of two, such that, upon completion of the cycling, millions
of copies of the DNA target are available for subsequent
analyses. Many different PCR approaches are available
(reviewed by Monis et al., 2005; Gasser, 2006), and those
applied to Eimeria are reviewed in the following.
4.4.1. ‘Fingerprinting’ techniques for the analysis of
genetic variation and definition of specific genetic markers
Such methods allow a genetic fingerprint of a parasite
sample to be produced. They rely on the screening of the
genome(s) for variation in sequence and/or organization,
and the data generated for species or populations can be
used to investigate specific identity, genetic diversity and/
or relationships. The advantage of such approaches is that
no prior sequence information is required about the
genomes of the parasites to be studied.
4.4.1.1. Amplified fragment length polymorphism
(AFLP). The method of AFLP is a high stringency
fingerprinting technique which relies on the selective
amplification of restriction fragments produced from
genomic DNA and their subsequent display by denaturing
gel electrophoretic analysis (Vos et al., 1995). There are
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
three main steps in the process: (a) total digestion of
genomic DNA and ligation of oligonucleotide adapters to
the resultant fragments; (b) selective amplification of
certain sets of restriction fragments; and (c) analysis by
gel electrophoresis of the amplified fragments (Vos et al.,
1995). The PCR amplification of step (b) is made possible
by using the adapter and restriction site sequence as the
target site for primer annealing. This process is made
selective by the choice of primers which extend into the
restriction fragments; only those fragments in which pri-
mer extensions match the nucleotides flanking the restric-
tion site will be amplified (Vos et al., 1995). Typically,
between 50 and 100 fragments are amplified, and these
are then separated by gel electrophoresis to create a
‘fingerprint’. This useful technique, while using highly
specific primer annealing and amplification, does not
require previous knowledge of the genome sequence (i.e.,
primers do not need to be specifically designed to target
the genome of the organism of interest).
AFLP has been used to derive polymorphic markers
for E. tenella for the construction of a genetic linkage map
(Shirley and Harvey, 2000). Different combinations of
primers were used on the same source material with
varying degrees of success; the best combinations yielded
10 polymorphic fragments per gel (Shirley and Harvey,
2000). Blake et al. (2003) examined a single (designated
Houghton) strain of E. maxima. Using a panel of nine E.
maxima samples and a range of primers, reproducible
AFLP fingerprints were consistently generated.
Various combinations of restriction enzymes and
primers should be evaluated to optimize the conditions
and number of amplified fragments generated; also high
quality and high molecular weight DNA is required. As
the primers employed are not designed to specifically
target the organism under investigation, the technique can
give spurious results if contamination is present in the
sample (Blake et al., 2003). Also, this technique cannot be
used to detect mixed-species infections, as multiple se-
quences can be hidden within bands of the same mole-
cular weight on electrophoretic gels.
4.4.1.2. Random amplification of polymorphic DNA
(RAPD) and sequence-characterized amplified regions
(SCARs). The random amplification of polymorphic
DNA (RAPD) or arbitrarily primed-polymerase chain
reaction (AP-PCR) (Welsh and McClelland, 1990;
Williams et al., 1990) relies on the amplification of
genomic DNA fragments using (usually) single primers
(∼ 10-mers) of arbitrary sequence, and subsequent elec-
trophoretic separation of the amplicons. The advantages
of RAPD include speed, simplicity, the relatively tiny
amounts of genomic DNA required and the ability to
597
screen the entire genome. Although RAPD is a useful
method, there may be problems with specificity and/or
reproducibility of results due to the low annealing tem-
peratures (i.e., 35–45 °C) frequently used in the PCR
(MacPherson and Gajadhar, 1993).
RAPD has been used to study variation within
species of Eimeria from chickens. Shirley and Bum-
stead (1994) examined seven strains of E. tenella of
varying biological phenotypes (including three estab-
lished laboratory strains, an egg-adapted, attenuated strain
and two precocious strains derived from these, as well as a
field isolate). Twenty-four arbitrary primers were used in
separate PCRs to amplify DNA. Amplicons were electro-
phoretically separated on agarose gels to display profiles.
Of 199 bands displayed, 4 (2%) were considered to be
variable among the seven samples examined, providing
strain-specific markers (Shirley and Bumstead, 1994).
Barta et al. (1998) employed RAPD to confirm the identity
of five strains of E. maxima. However, with the seven
arbitrary primers tested, these authors were not able to
identify sufficient unique bands to discern the relationships
among the strains. Some researchers have used the
calculation of similarity coefficients to gain a measurement
of degree of relatedness (Procunier et al., 1993; Johnston
and Fernando, 1995; Costa et al., 2001). This approach
employs a modification of Nei and Lei's (1979) original
analytical approach; the similarity coefficients depend on
the number of bands amplified from each of a pair of
species/strains and the number of shared bands (Procunier
et al., 1993; Johnston and Fernando, 1995). For example,
Procunier et al. (1993) found that two strains of E.
acervulina were less closely related than two of E. tenella,
having similarity coefficients of 61% and 98%, respec-
tively. RAPD data have also been promoted for investi-
gating evolutionary relationships among strains, but this
application is inappropriate, as bands in a profile represent
phenotypic characters (as they are separated based on size
alone and not sequence) rather than genetic characters
(which are linked to a DNA sequence).
Another use of RAPDs is for the identification of,
and discrimination among, species. The typical ap-
proach has been to test numerous random primers and
establish which ones result in unique patterns for each
species. MacPherson and Gajadhar (1993) were the first
to use this technique to distinguish among seven species
of Eimeria, one of which was from chickens (E.
tenella). Another early study (Procunier et al., 1993)
applied nine primers to six species of chicken Eimeria;
this approach could be used for the differentiation
among all species tested. More recently, studies have
confirmed the ability of RAPD techniques to distinguish
between Eimeria species from chickens (e.g., Johnston
598 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
and Fernando, 1995; Tsuji et al., 1997; Costa et al.,
2001; Fernandez et al., 2003a).
As RAPD is usually conducted under low-stringency
conditions (i.e., low annealing temperature), there can be
problems with the specificity and reproducibility of
results (Fernandez et al., 2003a,b). Also, this technique
is inappropriate for detecting mixed-species infections, as
multiple sequences can be hidden within co-migrating
bands on an electrophoretic gel (Fernandez et al., 2003b).
The main advantage of RAPD over other PCR-based
techniques is that primers are arbitrary in sequence,
requiring no previous knowledge of the genome sequence
(s) (MacPherson and Gajadhar, 1993; Procunier et al.,
1993; Johnston and Fernando, 1995; Fernandez et al.,
2003b). The technique is also rapid and relatively simple,
and requires relatively little genomic DNA template
(MacPherson and Gajadhar, 1993; Johnston and Fer-
nando, 1995).
A further advantage of RAPD is that it can be used to
define ‘sequence-characterized amplified regions’
(SCARs), which can represent species-specific or
strain-specific genetic markers. RAPD bands displayed
in an electrophoretic gel are excised, purified, cloned and
sequenced. Once the sequences have been determined,
they are called SCARs. Primers designed to them can be
used for targeted PCR-amplification (at high stringency)
from species or strains. Recently, this approach has been
applied to Eimeria of chickens (Fernandez et al., 2003a,
b). An initial RAPD-screening of samples of E. acer-
vulina, E. tenella and E. maxima using 150 different
primers resulted in 110 of these producing species-spe-
cific band profiles after electrophoresis. In addition to
species differentiation, numerous primers produced
strain-specific markers; higher variability was detected
among E. acervulina strains than either E. tenella or E.
maxima (Fernandez et al., 2003a). Species-specific
SCARs were defined for each of the seven species of
Eimeria and a primer set was designed for each (Fer-
nandez et al., 2003a). Each of these primer sets reliably
and reproducibly amplified DNA from the appropriate
species, resulting in a PCR product that appeared as a
single band on agarose gel, but not from heterologous
species (Fernandez et al., 2003a). In a subsequent study
(Fernandez et al., 2003b), the same research group deve-
loped a multiplex PCR assay based on SCAR markers.
Effective and specific amplification was achieved using
this multiplex PCR from as little as 1–5 pg of DNA
(corresponding to 2–8 sporulated oocysts) (Fernandez
et al., 2003b). In addition, the seven amplicons
representing individual species of Eimeria could be
readily distinguished based on their size on agarose gels
(Fernandez et al., 2003b). This PCR assay was tested on
33 strains, with at least two strains representing each of
the seven species. A product was amplified from each
sample, and no intra-specific variation in band size was
detectable (Fernandez et al., 2003b). More recently,
Fernandez et al. (2004) determined 151 different
SCARs (84 being species-specific and 67 having partial
specificity) representing the seven species of Eimeria
from chickens and established a SCAR database for
Eimeria to provide an in silico resource for other
researchers (see: http://puma.icb.usp.br/eimeriaScardb/).
By comparison with public gene sequence databases,
Fernandez et al. (2004) also showed that only 15 of the
SCARs had significant similarity to sequences in other
databases, suggesting that most SCARs had not been
identified previously and that they were likely to have
been derived from anonymous, non-coding DNA.
The development of a multiplex PCR for the simul-
taneous detection of all seven species of Eimeria from
chickens has been a very valuable technological advance
in diagnosis (Fernandez et al., 2003b). However, the
definition of SCAR-derived species-specific primers has
the disadvantage that separate primer pairs have to be
designed and tested for each species, and the multiplex
reaction required for the assay is relatively laborious to
initially set up.
4.4.2. PCR-based detection methods using genetic
markers in nuclear ribosomal DNA
In contrast to fingerprinting methods, PCR detection
assays rely on the amplification of particular regions of
genomic DNA using a defined set of oligonucleotide
primers. For example, Stucki et al. (1993) designed a
primer pair to amplify by PCR the 5S ribosomal RNA unit
of E. tenella. While this PCR could amplify from as few
as two oocysts of E. tenella, no product was amplified
from any of the five other Eimeria species tested (E.
acervulina, E. maxima, E. praecox and two species not
found in poultry). Similarly, Molloy et al. (1998) designed
primers to a sporozoite antigen gene (EASZ240/160).
This assay was specific for E. acervulina and did not
amplify product from any of the other six Eimeria species
from chickens. In both cases, an effective tool was deve-
loped for the identification of a single species of Eimeria,
but the approach had a limitation in that heterologous
species could not be detected. Other, more recent studies
focused on the use of the first and second internal trans-
cribed spacers (ITS-1 and ITS-2, respectively) of nuclear
ribosomal DNA (rDNA), which separate the ribosomal
genes. While the ribosomal genes themselves tend to be
conserved, these spacers are relatively heterogenous in
length and sequence among species, such that specific
primers may be designed to the flanking gene sequences.
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
Like other regions of nuclear rDNA, these spacers evolve
in a concerted fashion and are repetitive in nature (Elder
and Turner, 1995), making any PCR assay (based on their
use) sensitive.
Schnitzler et al. (1998, 1999) chose the ITS-1 as a
target for PCR. Initially, genus-specific primers to con-
served regions flanking the ITS-1 were used to amplify a
product from each of the seven species of Eimeria. The
amplicon from each species was then purified, cloned and
sequenced. Seven different primer pairs were designed to
unique sections of the ITS-1 sequence, one for each
species. Other workers (Lew et al., 2003; Su et al., 2003)
used a similar approach, employing genus-specific pri-
mers as a starting point, cloning and sequencing the ITS-1
region, and then designing species-specific primers. The
above studies employed one of two methods for the
detection of the amplicons produced. Typically, PCR
products underwent electrophoresis on ethidium bromide-
stained agarose gels, their sizes being displayed (Schnit-
zler et al., 1998; Lew et al., 2003; Su et al., 2003). While
agarose gels allow the resolution of a PCR product based
on size, they are limited in their ability to display sequence
variation among amplicons, unless it relates to a con-
siderable length difference. Schnitzler et al. (1999) also
used a paper chromatography hybridization assay (PCHA)
for the detection of amplicons. Briefly, the principle is that
oligonucleotide capture probes corresponding to the for-
ward primer and extending into the expected product to
30 bp being bound to a ‘comb’; the denatured biotinylated
PCR product is then incubated with the labelled comb,
permitting binding between complementary strands. A
positive reaction is indicated by a purple dot appearing on
the comb. Thus, the PCHA method gives a simple positive/
negative test result, but does not provide any information
about genetic variation.
In these studies (Schnitzler et al., 1998; Lew et al.,
2003; Su et al., 2003), each primer pair amplified
specifically and reproducibly from the appropriate
species but not from heterologous species. However,
no evaluation of all primers in a single, multiplex
reaction was reported. Hence, using these PCR-based
assays, each sample must be subjected to multiple PCR
reactions to detect which species are present, which is
laborious and time consuming. If a single set of primers
could simultaneously amplify from each of the seven
species of Eimeria, producing specific amplicons
which are clearly distinguishable in size among all
species, then much labour could be avoided and the
assay would be less expensive to perform. In addition,
the electrophoretic methods used (i.e., agarose gels and
PCHA) are unable to detect sequence variability in ITS-
1 amplicons.
599
4.4.3. PCR-coupled manual and semi-automated elec-
trophoretic methods for the diagnosis and/or analysis
of genetic variation within and among species of
Eimeria
For Eimeria species of chickens, the direct sequencing
of ITS-1 or ITS-2 amplicons is not feasible due to se-
quence heterogeneity within individual isolates (Schnit-
zler et al., 1998, 1999; Woods et al., 2000a,b; Lew et al.,
2003). Thus, sequencing of cloned amplicons has been
the most commonly used approach to define species-
specific genetic markers in these spacers. However, the
sequencing of cloned amplicons (representing the internal
transcribed spacers) has limitations in that it does not
allow and accurate characterization of the sequence vari-
ability and because PCR-induced artefacts may lead to
erroneous sequence data. Recently, electrophoretic appro-
aches have been utilized for the direct display of sequence
heterogeneity within and among amplicons (reviewed by
Gasser, 2006) and have been applied to the internal tran-
scribed spacers of Eimeria.
For instance, Woods et al. (2000b) employed PCR-
based RFLP analysis on high-resolution electrophoretic
gels for the initial characterization of sequence variation in
the ITS-2 within and among samples representing six
different species of Eimeria from chickens from Australia,
in order to assess whether this rDNA region would pro-
vide genetic markers for their specific identification. The
ITS-2 was amplified from genomic DNA by PCR using a
radiolabelled primer set, the forward primer (WW2) being
Eimeria-specific and the reverse one (WW4R) being
Eimeriidae-specific, digested separately with three re-
striction endonucleases (CfoI, Sau3AI or TaqI) and the
fragments separated by denaturing gel electrophoresis.
The PCR products amplified separately from isolates
representing six species (E. acervulina, E. brunetti, E.
maxima, E. necatrix, E. praecox and E tenella) varied in
size from ∼370 to 620 bp on agarose gels, with diffe-
rences in size and number of bands among species but
limited variation within a species. The PCR-RFLP ana-
lysis of ITS-2 amplicons on denaturing gels gave cha-
racteristic profiles for individual species (except for minor
variation in profiles within some species), which indicated
that ITS-2 contained useful genetic markers for the iden-
tification of six Eimeria species occurring in Australia.
Subsequently, Woods et al. (2000a) employed denaturing
gel electrophoresis and single strand conformation poly-
morphism (SSCP) for the direct analysis of radiolabelled
ITS-1 and ITS-2 amplicons from Eimeria species from
chickens. While the former electrophoretic approach
separates single-stranded molecules based on size alone,
the latter has the major advantage that it separates them
based on both size and sequence (see Gasser and Zhu,
600 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603

1999). Both electrophoretic approaches allowed the
unequivocal identification and delineation of all six
species tested; in addition, the SSCP allowed the detection
of population variation within E. acervulina not detect-
able by denaturing gel electrophoresis (Woods et al.,
2000a). While successful at delineating species, both
electrophoretic approaches required the use of radiola-
belled primers in the PCR and that controls be run
simultaneously as reference standards.
Due to the increased demands for high-speed, high
throughput diagnosis, genetic analysis and data handling-
analysis in silico, there have been substantial enhance-
ments in the performance and efficiency of automated
electrophoretic procedures. Previous studies (Woods et al.,
2000a,b) were extended to establish semi-automated
electrophoretic approaches for the specific identification
and differentiation of all seven currently recognized
species of Eimeria infecting chickens (Gasser et al.,
2001, 2005). The ITS-2 was PCR-amplified from all
seven species using the original set of oligonucleotide
primers (WW2 and WW4R), of which WW2 was labelled
fluorescently. Following the PCR, the amplicons were
diluted (1/30) in a specific (denaturing) loading buffer,
heat-denatured and subjected directly to slab gel (Gasser et
al., 2001) or capillary electrophoresis (CE) (Gasser et al.,
2005; summarised in Fig. 1). The chromatograms cap-
tured were stored electronically and then analysed using a
specific software program. Using well-defined control
DNA samples representing monospecific lines of Ei-
meria, particular peaks in the chromatograms were de-
fined for the identification of each of the seven species and
for their unequivocal differentation. Some variation in the
positions of ‘minor’ peaks in the chromatograms (re-
flecting population variation) was detectable for some
species, but did not interfere with specific identification.
Unpublished findings have shown that CE can effectively
detect one nucleotide deletion/insertion event for ampli-
cons of up to 300 bp and two such events for a fragment
length of up to 600 bp (Rob Skinner, personal
communication). To test the effectiveness of the approach,
samples from chickens with naturally acquired infections
of multiple Eimeria species were examined, and the

Fig. 1. A polymerase chain reaction (PCR)-coupled capillary electrophoretic approach has been developed for the identification of seven currently
recognized species of Eimeria infecting chickens (see Gasser et al., 2005). Total genomic DNA is released from samples (e.g., oocysts or gut content)
using glass beads and proteinase K/sodium dodecyl-sulphate (SDS)-digestion, followed by purification over mini-columns (step 1). The second
internal transcribed spacer (ITS-2) of ribosomal DNA is PCR-amplified, using a single set of oligonucleotide primers (one of which is fluorescently
labelled), from genomic DNA from any of the seven species (step 2). Amplicons are individually diluted in a loading buffer (usually 1/30 dilution),
arrayed in a microtitration plate, heat denatured and then subjected to automated capillary electrophoresis in a MegaBACE™ 1000 (Amersham). The
chromatograms captured are stored electronically and then analysed using MegaBACE™ Fragment Profiler software (step 3). Using control DNA
samples representing monospecific lines of Eimeria, specific peaks in the chromatograms have been defined for the unequivocal identification of
each of the seven species (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella are represented by A, B, M, μ, N, P
and T, respectively) and their differentiation (step 4).
 G.M. Morris, R.B. Gasser / Biotechnology Advances 24 (2006) 590–603
species compositions were readily determined by com-
parison with monospecific reference samples (Gasser et
al., 2005). Such an automated approach should find
applicability as a tool for the quality control of live Ei-
meria vaccines (i.e., verifying their species composition),
the monitoring of coccidiosis outbreaks, post-mortem
diagnosis, and the high throughput analysis of oocyst
samples (from faeces or the environment) for epidemio-
logical surveys.
Clearly, the CE approach has major advantages over
manual electrophoretic techniques, mainly in relation to
electrophoresis time, sample through-put, and data
storage and analysis capacities. There is no need for the
use of ethidium bromide or radionucleotides, or the
pouring, handling and/or exposure of electrophoretic gels,
thus substantially reducing overall time and cost. While
the CE equipment is expensive, any ‘genomics’ or se-
quencing facility can provide the service required for the
analysis of amplicons, once the electrophoretic conditions
have been optimized. Other advantages are that chroma-
tograms representing different samples, run on different
gels and different days, can be scored automatically and
stored electronically in a spreadsheet format, and can be
retrieved at any time point for comparative analyses. The
CE approach (Fig. 1) is thus suited for the screening of
large numbers of samples and, although qualitative, is
currently less expensive to carry out than, for example,
real-time PCR, and has the added advantages of being
able to detect genetic variability.
5. Concluding remarks
Biotechnological advances have been achieved in the
control of coccidiosis, particularly through the develop-
ment of live vaccines and molecular-diagnostic methods.
Progress in this area is of major relevance because of the
serious problems with resistance in Eimeria populations
against anticoccidial compounds (due to their excessive,
preventative use and the rapid generation times of Ei-
meria) and residue problems in chicken meat, eggs and
the environment. Given these key issues, the specific
diagnosis of Eimeria infections in chickens is clearly
central to a better understanding of the epidemiology and
dynamics of disease in intensive and extensive chicken
establishments, which underpins the effective prevention
and control of coccidiosis. Although some representatives
of the poultry industry may argue that “specific diagnosis”
represents an “academic exercise”, is costly and not of a
practical value, recent progress demonstrates clearly that
some PCR-based methods utilizing genetic markers in
nuclear ribosomal DNA provide rapid and powerful
complementary diagnostic and/or analytical tools. In par-
601
ticular, the PCR-coupled CE approach provides a plat-
form for high throughput and high resolution diagnosis
and genetic analysis. While, in the future, the real-time
PCR (using probes specific to individual species of Ei-
meria) is likely to become less expensive than is currently
the case, such that an inexpensive quantitative assay could
be developed, such an assay would not have the analytical
capacity of the PCR-coupled CE for the detection of
genetic variation. Currently, the CE approach is affordable
to the poultry producer and industry, as it employs only a
single set of primers in a standard PCR for all recognized
species of Eimeria. Given its advantages, the CE approach
is now being employed routinely to conduct epidemio-
logical surveys and to investigate, for the first time, the
abundance/intensity and dynamics of Eimeria infections
in selected poultry establishments in Australia, thus
logically complementing various prevention, vaccination
and control programs.
Acknowledgements
Funding support from the Australian Poultry Cooper-
ative Research Centre (CRC) is gratefully acknowledged.
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