Veterinary Parasitology 189 (2012) 65–74

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

Advances in diagnosis of protozoan diseases

T. de Waal ∗
School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

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

Keywords: Microscopy still remains the gold standard procedure for the diagnosis of many proto-
Apicomplexa zoan infections in animals, but the specific identification requires skilled and experienced
LAMP personnel. Immunoassays, detecting antibodies or specific protozoan antigens, have been
Microscopy
developed but often lack sensitivity and specificity due to close relationship between many
PCR
Serology
protozoa. Recent research has focussed almost exclusively on molecular based techniques
for the identification and quantification of parasite DNA in samples. Opinion differ on most
appropriate targets to use and there are very few diagnostic kits available making compar-
ison between laboratories difficult. Future research needs to focus on robust, cheap field
diagnostic assays.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction 2. Laboratory diagnostic methods for protozoan

The improvement of the microscope from a novelty to a
scientific tool by the Dutch scientist Antony van Leeuwen-
hoek in the 17th century has led to pioneering discoveries
concerning protozoa and their role as causative agents
of disease. The effective control and treatment of proto-
zoan diseases requires rapid, reliable and highly sensitive
diagnostic tests, in order to diagnose infections, as well
as to monitor the therapeutic and prophylactic protocols.
Today, microscopy is still widely used for the diagnosis
of protozoan infections in animals. Recent developments
in molecular biology have opened many new avenues for
the development of improved diagnostics tools for the
detection of protozoa. This aim of this short review is
to highlight the progression of the technologies and how
they have improved the diagnosis of protozoan diseases
in sheep.
∗ Corresponding author.
E-mail address: theo.dewaal@ucd.ie
infections
2.1. Classic diagnostic techniques
2.1.1. Microscopy
The most unequivocally diagnosis of protozoan infec-
tions is by demonstration of the organism in the blood, bone
marrow, cerebrospinal fluid, faeces or urine. The simplest
method of microscopic examination is the examination of
smears. In the case of protozoa occurring in the circulation,
blood smears are prepared and stained. The Romanofsky-
type stains usually give best results and can be routinely
applied to diagnose babesiosis, theileriosis or trypanoso-
mosis (Garcia, 1999).
In the case of gastrointestinal protozoa, the simplest
technique is a direct faecal smear with or without fur-
ther staining (Garcia, 1999). A direct saline smear of a
small amount of faeces mixed with saline is prepared on a
microscope slide. This is often used for the identification of
trophozoite stages of Giardia, Trichomonas and Balantidium.
The advantage is that the movement of these organisms can
be observed which aid in the identification. Direct smears
can also be used to identify the cyst stages of many of these
parasites. Further staining is often used to improve the
sensitivity of this technique. The greatest disadvantage of

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66 T. de Waal / Veterinary Parasitology 189 (2012) 65–74
Table 1
Immunodiagnostic techniques available for laboratory diagnosis of protozoa.
Test Protozoan genera for which can be applied
Complement fixation test Babesia, Theileria, Toxoplasma, Trypanosoma
Sabin–Feldman Dye test Toxoplasma
Indirect fluorescent antibody test Babesia, Theileria, Toxoplasma, Neospora
Radioimmunoassay
ELISA Babesia, Theileria, Toxoplasma, Neospora, Trypanosoma,
Cryptosporidium
Competition-inhibition ELISA Babesia, Theileria, Trypanosoma
Latex agglutination test Toxoplasma
Trypanosoma
Antigen detection ELISA Cryptosporidium, Giardia
smears is its lack of sensitivity; to overcome this, methods
for concentrating protozoa from a larger volume of start-
ing material before microscopic examination have been
developed. In the case of gastrointestinal protozoa, faecal
flotation is commonly used to concentrate cysts/oocysts in
faeces. This method is based on the difference in specific
gravity of (oo)cysts and faecal debris and by using specific
flotation solutions allowing the cysts to float to the surface
of the solution and the larger particles of faecal debris sink
to the bottom, thus ‘concentrating’ the cysts on the surface
of the solution. The most common flotation solutions used
to concentrate protozoan cysts are Sheather’s solution and
ZnSO4 (Garcia, 1999).
Thick blood smear allows examination of a slightly
larger amount of blood than a thin blood smear and is often
used in the diagnosis of Babesia infections, while a buffy
coat method is another concentration technique often used
for the detection of trypanosomes in blood samples.
2.1.2. Indirect diagnostic methods
If organisms occur at densities below the sensitivity of
the direct method employed or cannot be directly demon-
strated in a biological sample due to the life cycle in the host
false negative results may be encountered. Numerous sero-
logical tests have been developed to indirectly diagnose
infections.
2.1.2.1. Immunodiagnosis – antibody detection. Tests com-
monly used to detect the presence of antibodies against
a specific protozoan include the complement fixation test
(CFT), the immunodiffusion (ID), the indirect haemagglu-
tination (IHA), the latex agglutination (LA), the indirect
fluorescent antidody test (IFAT), the radio-immunoassay
(RAI) and the enzyme-linked immunosorbent assay (ELISA)
(Table 1). A drawback of serodiagnosis is the fact that
antibodies persist for long time, even after elimination of
the parasite, therefore a positive result does not necessar-
ily indicate the present parasitological status of the host.
Moreover, serology is not useful to diagnose acute infec-
tions. Cross reactions are also often encountered between
closely related parasites resulting in false positive outcome
such as trypanosomes. With the development of molec-
ular techniques, it has been possible to develop test that
are based on specific subunit proteins/antigens given much
greater specificity to these tests for Babesia, Theileria and
Trypanosoma (Katz et al., 2000).
References
Warren and Sabin (1942), Böse et al. (1995), Herr
et al. (1985)
Sabin and Feldman (1948)
Böse et al. (1995), Seefeldt et al. (1989)
Kahl et al. (1982)
OIE (2010), Bjorkman et al. (1999), Buxton (1998),
Seefeldt et al. (1989), Von Blumröder et al. (2004),
Smith (2008)
Katz et al. (2000).
Buxton (1998), Seefeldt et al. (1989)
Asonganyi et al. (1998)
Weitzel et al. (2006)
2.1.2.2. Antigen detection. An alternative to improve diag-
nosis is to specifically detect parasite antigens, rather than
host antibodies against the parasite. Currently, there are
several antigen detection tests available for in vitro diag-
nosis of Giardia and Cryptosporidium in faecal samples
(Table 1). A drawback of many of the diagnostic assays
is the lack of standardised reagents resulting in variation
in results between laboratories. However, more and more
protozoan serological and antigen assays are becoming
commercially available.
2.2. Nucleic acid-based diagnostics
2.2.1. Multilocus enzyme electrophoresis
Multilocus enzyme electrophoresis is a method for
characterizing organisms by the relative mobilities under
electrophoresis of a large number of intracellular enzymes.
Differences in the electrostatic charge and size between
homologous enzymes as a result of the underlying varia-
tion in the originally transcribed DNA sequence will affect
its electrophoretic mobility. Thus, it is possible to relate
mobility differences to different alleles at the gene locus for
the enzyme in question. Multilocus enzyme electrophore-
sis has been used to characterize Trypanosoma isolates
(Barnabé et al., 2000) and Eimeria spp. (Shirley, 1975).
Multilocus enzyme electrophoresis has many drawbacks;
strains with the same enzyme phenotype may in fact have
distinct amino acid sequences, the degree of relationship
between different phenotypes is not known, putative het-
erozygous phenotypes are difficult to interpret, it is time
consuming and expensive and requires large volume of
parasite material.
2.2.2. Southern blot technique
In the Southern blot technique, DNA fragments are
digested using one or more restriction enzymes and sepa-
rated by electrophoresis before being transferred (blotted)
onto membrane filters and hybridized with complemen-
tary (radio) labeled probes (Southern, 1975). Methods
for non-radioactive labelling of DNA probes have also
been developed and include the incorporation of reporter
molecules, such as biotin (Murasugi and Wallace, 1984),
acetylaminofluorenzyl modified guanosine (Tchen et al.,
1984) and sulphonated cytidine (Poverenny et al., 1979).
Detection of these molecules is with an appropriate anti-
body or, in the case of biotin, with avidin or strepavidin
 T. de Waal / Veterinary Parasitology 189 (2012) 65–74 67

Table 2
Real-time PCR detection of protozoan parasites.

Detection chemistry Examples of protozoa for References

which can be applied

Intercalating dyes (SYBR Green I) Fluorescence when bound to Cryptosporidium parvum Widmer et al. (2004)
dsDNA, but not when free in Leishmania Nicolas et al. (2002)
solution Trypanosoma brucei Becker et al., 2004

TaqMan probes Fluorescence following hydrolysis by Taq Cryptosporidium Higgins et al. (2001),
DNA polymerase Keegan et al. (2003)
Giardia Bertrand et al. (2004)
Toxoplasma gondii Jauregui et al. (2001)
Theileria parva Papli et al. (2011)

Fluorescence resonance-energy-transfer
(FRET) assay
Energy transfer between donor fluorophore and Toxoplasma gondii Simon et al. (2004)
reporter fluorophore at 3 and 5 ends,
respectively, of 2 different probes. Fluorescence
is detected only when the probes hybridize
adjacent to each other on the target DNA. Cryptosporidium parvum Limor et al. (2002)

coupled to a colorimetric, fluorimetric or chemilumines-
cent signal. Direct cross-linking of probes to enzymes
which act as signal generators has also been described
(Renz and Kurz, 1984). DNA probes have been developed
for the detection of various protozoa in both mammalian
hosts and insect/tick vector, including Babesia spp., Thei-
leria spp. and Trypanosoma spp. (McLaughlin et al., 1986;
Posnett and Ambrosio, 1989, 1991; Petchpoo et al., 1992;
Hodgson et al., 1992). The first nucleic acid-based detec-
tion and characterisation of trypanosomes were done by
using the genes coding for trypanosome variable surface
glycoproteins (Williams et al., 1982; Majiwa et al., 1985b;
Majiwa and Webster, 1987), which can detect the parasite
in the tsetse fly vector (Kukla et al., 1987; Gibson et al.,
1988). Ellis and Bumstead (1990) also developed probes
that could distinguish between various Eimeria spp. A lim-
itation of this technique is that an appropriate probe must
be designed to hybridise to the digested DNA fragments
and a rather large number of organisms to process.
2.2.3. PCR
Development of the polymerase chain reaction (PCR) in
1985 has revolutionised the diagnosis of infectious diseases
in general (Saiki et al., 1985, 1988). With PCR, a specific
DNA fragment from complex DNA samples can be ampli-
fied resulting in many millions of copies of the target DNA
molecule. The standard method requires a DNA template,
containing the region to be amplified and two oligonu-
cleotide primers flanking the target region. PCR products
can then be visualized by separating them electrophorec-
tically according to size on agarose gels. Since the original
description various modifications has been developed to
further increase the sensitivity and specificity of the ampli-
fication procedure, such as nested PCR, in which the PCR
product is subjected to a second round of amplification
with a second pair of oligonucleotide primers located inter-
nally from the first pair (Dieffenbach et al., 1993). The
reverse-line blot assay, which allows for the identification
of novel genotypes or species and also allows for the detec-
tion of mixed infections has been developed for Babesia
and Theileria infections (Gubbels et al., 1999; Nagore et al.,
2004).
The next enhancement of this technology came with
the development of real-time amplification. The primary
advantage of RT-PCR over conventional PCR is that it pro-
vides for high-throughput analysis in a closed system,
thus eliminating the problems of cross-contamination. This
method can also be used to quantify by exploiting the
proportional relationship between the threshold cycle, at
which exponential amplification is detected and the start-
ing number of the copies of the target fragment. Various
RT-PCR detection chemistries have been developed and
applied in the detection of protozoa (Table 2).
2.2.4. LAMP
Alternative DNA amplification, such as loop mediated
isothermal amplification (LAMP) has been applied to pro-
tozoa (Alhassan et al., 2007; Karanis et al., 2007; Njiru et al.,
2008; Guan et al., 2008; Karanis and Ongerth, 2009). In
this method, six different primers, specifically designed
to recognise eight distinct regions on a target gene, with
amplification only occurring if all the primers bind and
form a product. Unlike PCR, LAMP is carried out at a temper-
ature range of 60–65 ◦ C eliminating the need of a thermal
cycler. In addition, the reaction can be carried out without
the need of DNA extraction.
3. Advancement of detection methods in selected
protozoan diseases
3.1. Piroplasmosis
Piroplasmoses are tick borne infections caused by
intra-erythrocytic protozoan parasites belonging to sev-
eral Babesia or Theileria species, infecting a wide range
of domestic animals worldwide. Theileria lestoquardi, Thei-
leria ovis, Theileria separata, Babesia ovis, Babesia motasi,
Babesia crassa have been identified from sheep (Uilenberg,
2006). B. ovis are usually regarded as the most important
disease agent. In addition, several novel Babesia and Thei-
leria (Theileria uilenbergi and Theileria luwenshuni) species
were isolated from naturally infected sheep in China, where
it cause severe and often lethal disease (Yin et al., 2007;
Guan et al., 2010). In endemic areas, they are of significant
68 T. de Waal / Veterinary Parasitology 189 (2012) 65–74
financial significance, due to production losses and
increased mortality rate.
Piroplasmosis can be diagnosed by the examination
of peripheral blood smears or smears from visceral
organs (brain/kidney/lung/lymph nodes) stained with
Romanovsky-type staining methods, such as the Giemsa
stain. In carrier animals, it is quite difficult, if not impos-
sible, to demonstrate parasites, as the number of parasites
fall below detectable levels soon after the acute stages of
the disease. While it is possible to differentiate the differ-
ent Babesia species based on their morphology, this is rarely
possible in the case of Theileria infections.
A number of serological assays are available to detect
antibodies in carrier animals. The drawback of serological
assays, as described above, is that presence of antibod-
ies only confirms exposure to the parasite in questions
and does not indicate acute infection, nor confirms the
carrier state. Moreover, many of these tests have been
developed in-house at specific laboratories, hence very few
are commercially available, with standardised antigens and
test procedures, thus making interpretation and compari-
son between regions difficult. However, serological assays
are commonly used for testing animals as requirement
for international trade (OIE, 2010). A CFT has been devel-
oped to detect antibodies against a variety of Babesia and
Theileria parasites (Table 1). Titres usually rise soon after
infection, but also decrease rapidly in carrier animals. Cross
reactions are common between closely related species.
The immunofluorescent antibody test using smears of
Babesia/Theileria as antigen is still widely used and more
sensitive than the CFT (Böse et al., 1995). ELISA test have
been developed for various Babesia and Theileria parasites.
Antigens can be crude lysates obtained from infected ery-
throcytes, soluble extracts from in vitro cultures or subunit
antigens produced in vitro (Katz et al., 2000).
DNA probes have been developed to detect Babesia DNA
in infected animals, based primarily on sequences of the
18S rDNA gene (Böse et al., 1995). To increase the sensi-
tivity of these techniques PCR reaction is used to amplify
specific target sequences (Böse et al., 1995). A reverse-
line blot assay has been developed for the simultaneous
identification of animals carrying different species of Thei-
leria and/or Babesia simultaneously. The assay employs one
set of primers that specifically amplify the rRNA gene V4
hypervariable region of all Babesia and Theileria species.
The PCR product obtained are then hybridised to a nitro-
cellulose membrane, onto which different species-specific
oligonucleotide probes are covalently linked (Sparagano
and Jongejan, 1999). The assay has been used in epidemi-
ological surveys in various countries (Almeria et al., 2002;
Niu et al., 2009).
3.2. Trypanosoma infections
Trypanosoma generally cause chronic infections in
sheep and are transmitted by haematophagus insects
either biologically with Glossina spp or mechanically
by Tabanids or Stomoxys spp. Sheep are infected by
Trypanosoma brucei, Trypanosoma congolense and Try-
panosoma vivax with the latter being the most preva-
lent infection in many parts of Africa. Trypanosoma
melophagium is a non-pathogenic parasite of sheep trans-
mitted by Melophagus ovinus with a worldwide distribution
(Martinkovic et al., 2012).
Trypanosomes can be demonstrated microscopically in
infected animals by examining fresh or fixed and stained
smears prepared from blood or lymph nodes. However,
sensitivity of this method is quite low and can only detect
>104 parasites/ml of blood. Although fixed and stained
blood/lymph node smears are useful for the specific iden-
tification of trypanosomes to the subgenus level, based on
morphology and morphometry, its sensitivity is lower than
that of fresh blood. Various concentration techniques have
been developed to increase the sensitivity of microscopic
examination, such as the haematocrit centrifugation tech-
nique (also known as the Woo test) with a sensitivity of
∼103 parasites/ml of blood.
Because of the low concentration of parasites in biolog-
ical samples, a widely used method is the in vivo culture of
the parasites by the intraperitoneal inoculation of the sam-
ples into mice. The method is often used for the diagnosis
of Trypanosoma evansi infections, a parasite which is par-
ticularly virulent in the mice with results being obtained
within 3–5 days. The success of this technique is variable
with other Trypanosoma species. Because of the lag time
in diagnosis and the cost and ethical considerations, this
technique is not used for the routine diagnosis.
Use of serological techniques is useful for epidemiologi-
cal studies. However, considerable antigenic cross-reaction
occurs between Trypanosoma species, and no serologically
technique is available that will confirm the identification of
species. The common tests available include the agglutina-
tion, the card agglutination, the CFT, IFAT and the ELISA. As
indicated for piroplasms, the production and standardisa-
tion of antigens used in these assays remains a constraint.
Since trypanosomes are intravascular parasites and
they would release many components, including spe-
cific antigens into the blood stream of the infected host,
the detection of these antigens has been investigated. A
sandwich-ELISA using a series of monoclonal antibodies
developed in the late 1980s (Nantulya et al., 1987), which
could detect three specific subgenera with high sensitivity
and specificity were developed and subsequently applied
in the field. However, further field validations showed that
these reagents were less sensitive with some cross reaction
occurring and maybe ascribed to the fact that hosts con-
tain multiple infections causing errors in the test and this
approach has now been abandoned (Eisler et al., 1998).
The first nucleic acid-based detection and characterisa-
tion of trypanosomes was based on probes for the variable
surface glycoprotein genes (Adams and Hamilton, 2008).
No cross-hybridisation occurred with other trypanosome
species and it was even possible to distinguish between dif-
ferent groups of T. congolense. The ‘first generation’ of PCR
tests relied on species-specific hybridisation probes based
on satellite DNA sequences offering increased sensitivity
and specificity over other techniques (Majiwa et al., 1985a;
Majiwa and Webster, 1987). However, since this test would
require a panel of probes to distinguish between the dif-
ferent species in a field sample, it is expensive and several
approaches have since been investigated to set up multi-
specific diagnosis within a single reaction. One approach is
 T. de Waal / Veterinary Parasitology 189 (2012) 65–74
Table 3
Sensitivity of various diagnostic techniques for Cryptosporidium.
Technique
Unconcentrated faecal smears
Faecal smear following concentration
FITC-mAb staining
Antigen capture ELISA
PCR based methods
the amplification of the ITS-1 region which enables simul-
taneous detection of seven Trypanosomes, even in mixed
infections (Desquesnes et al., 2001). However, this test
lacked sensitivity, especially for T. vivax. Cox et al. (2005)
increased the sensitivity of this technique by developing
nested PCR strategies. Other studies have used generic
primers in a semi-nested PCR assay to amplify the variable
region of 18S rDNA gene followed by restriction enzymatic
digestion. With this restriction fragment polymorphism
approach it was possible to distinguish between the impor-
tant trypanosome species infecting cattle even with mixed
infections (Geysen et al., 2003; Delespaux et al., 2003).
With the use of species-specific PCR tests, it is now possible
to identify the 11 tsetse-transmitted trypanosome species
and subgroups for which there are available primers.
A fluorescent fragment length barcoding method has
also been described, which was able to detect and dis-
tinguish trypanosomes (with the exception of members
of the subgenus Trypanozoon) in both laboratory and field
experiments (Adams and Hamilton, 2008; Hamilton et al.,
2008) and was reported to be more sensitive than the ITS
method. The big drawback of this technique is its cost and
the requirement for specialised equipment.
A promising development is the application of the LAMP
method for Trypanosoma detection. Recently this technique
has been adapted to detect African trypanosomes. A num-
ber of primers have been described for the detection of T.
brucei, T. congolense, T. vivax, Trypanosoma gambiense and
T. evansi (Kuboki et al., 2003; Njiru et al., 2008; Thekisoe
et al., 2007).
3.3. Cryptosporidium infections
Cryptosporidium is an ubiquitous small coccidian par-
asite infecting a wide range of mammals, birds, reptiles
and fish. The parasite has a direct life cycle and is gener-
ally transmitted through the faecal oral route. The oocysts
are highly resistant in the environment and most chemi-
cal disinfectants have no effect. It is also recognised as an
important waterborne infection. Currently more than 20
species are recognised, with many more genotypes being
identified. The majority of these species tend to be host
specific and are not considered pathogenic to the immuno-
competent hosts. The exception is Cryptosporidium parvum,
which infects a wide variety of domestic and wild animals,
including humans, and is often the primary cause of diar-
rhoea in newborn animals. Among domestic ruminants,
newborn kids are the most susceptible species, followed
by calves and lambs. Although some studies have indi-
cated that C. parvum occurs less frequently in sheep, others
have shown that the failure to detect C. parvum may have
69
Sensitivity (oocysts per g of faeces) References
106 Webster et al. (1996)
1 × 104 –5 × 104 Weber et al. (1991)
1 × 103 Webster et al. (1996)
3 × 105 –1 × 106 Smith (2008)
<100 Webster et al. (1996)
been due to the preferential amplification of the dominant
species in mixed infections (Xiao, 2010).
In the 1970s Cryptosporidium was first recognized as
an important aetiological agent in newborn calf diar-
rhoea complex (Pohlenz et al., 1978). Initially, diagnosis
of the infection was based on demonstrating the organ-
ism in histological sections. Henriksen and Pohlenz (1981)
investigated differential staining of the organism in faecal
smears, by using techniques routinely used in the bacterio-
logical laboratory and discovered that acid-fast techniques
were suitable differentiating stains. The Ziehl–Neelson
staining technique was the first staining technique to be
used routinely. The technique is based on the principle that
oocysts can be stained with carbol-fuchsin and retain the
dye during the decolourising step with acid alcohol. Var-
ious other direct staining method have been developed,
such as Auramine-O (Casemore et al., 1985); DMSO carbol-
fuchsin (Pohjola, 1984); Kinyoun (Ma and Soave, 1983)
and safranin-methylene blue (Baxby et al., 1984). Direct
staining is still widely used today for the demonstration of
oocysts in faecal matter. However, generally the sensitiv-
ity of these techniques is low, hence, due to their small size
and paucity in some samples, oocysts may be overlooked
or confused with yeast cells (Table 3). The use of fluores-
cently labelled monoclonal antibodies directed against the
oocysts wall of Cryptosporidium oocysts has been reported
to achieve higher specificities and sensitivities (Jex et al.,
2008). Commercially FITC-mAbs, directed against Cryp-
tosporidium oocyst wall, are available and routinely used for
the detection and enumeration of Cryptosporidium oocysts
in faecal and environmental samples (Smith, 2008).
Indirect methods to detect Cryptosporidium antigens in
faecal samples have also been developed and a number of
them are available commercially in an ELISA format. These
copro-antigen assays have been developed for detecting
C. parvum antigens in faeces, although when applied to
animal faecal samples results can be variable and some-
times less sensitive than routine microscopic approaches
(Johnston et al., 2003).
Given the limitation of these staining techniques and
inability to discriminate between species or genotypes,
various molecular methods have been developed. Fluo-
rescent labelled oligonucleotide probes targeting variable
regions of the ribosomal RNA can be used to detect oocysts
in environmental samples (Smith, 2008). These probes,
however, do not distinguish among species and geno-
types. This has lead to the development of various PCR
and nested PCR approaches targeting different genetic loci.
The most common loci used for the specific identification
of Cryptosporidium is the 18s ss rRNA gene; Cryptosporid-
ium oocysts wall protein (cowp), 70 kDa heat shock protein
(HSP70) and 60 kDa glycoprotein (gp60) (Smith, 2008).
70 T. de Waal / Veterinary Parasitology 189 (2012) 65–74
Specific enzymatic digestion of the products (fragment
polymorphism) or sequencing is then used to identify
species or genotypes. Boulter-Bitzer et al. (2007) reviewed
additional genetic markers with potential for diagnosis and
population genetic studies.
Real-time PCR has also been developed to quantify and
differentiate species and genotypes of Cryptosporidium in
animal, human and environmental samples (Monis and
Giglio, 2006). Mini- and microsatellite typing and gp60
sequencing have been used to subtype C. parvum and Cryp-
tosporidium hominis (Smith, 2008). Studies in recent years
have identified a number of Cryptosporidium species and
genotypes in sheep faeces. The most frequently described
species, apart from C. parvum, include Cryptosporidium
bovis, Cryptosporidium xiaoi (=C. bovis-like genotype) and
Cryptosporidium ubiquitum (=C. cervine genotype) (Ryan
et al., 2005; Mueller-Doblies et al., 2008; Robertson et al.,
2010).
3.4. Coccidian infections
Eimeria spp. are apicomplexan protozoan parasites that
infect a wide variety of domestic animals. Infection with
Eimeria spp. in sheep can cause either clinical (diarrhoea)
or subclinical disease (poor weight gain) and often is an
infection of significant economic importance especially in
intensively reared sheep (Chartier and Paraud, 2012). More
than 15 species of Eimeria have been described in sheep and
mixed infection often occurs. Eimeria ovinoidalis is consid-
ered, by far, to be the most pathogenic species (Catchpole
et al., 1976). The pathogenic effect of coccidia is often
further aggravated by other pathogenic agents, such as
gastro-intestinal nematodes, viruses or bacteria. Coccidio-
sis is diagnosed by demonstrating presence of the oocysts
during microscopic examination of faecal samples follow-
ing sugar/salt concentration, in conjunction with clinical
signs and the typical macroscopic lesions and location of
these lesions seen during post mortem examination.
Species identification is based on the morphology and
morphometrics of sporulated oocysts. Identification is
based on the size, shape and presence of characteristic ele-
ments, such as the polar cap, the micropyle, the colour
characteristic of the oocysts wall, the number of sporocysts
and sporozoites and the presence or absence of oocysts and
sporocyst residual bodies. Even then, diagnosis can be dif-
ficult and would require an expert to differentiate between
species.
Compared to many of the other protozoan diseases,
comparatively little research has been done on alterna-
tive diagnostic methods. Most research has been done on
improving the diagnosis on Eimeria spp. infecting poultry.
Shirley (1975) was the first to use a molecular biologi-
cal approach to differentiate Eimeria species on the basis
of isoenzyme patterns of oocysts by starch block elec-
trophoresis. Ellis and Bumstead (1990) were among the
first to demonstrate that rRNA and rDNA probes could
be used to identify individual species through charac-
teristic restriction fragment patterns. PCR methods have
been developed to detect Eimeria spp. by using ITS-1
or ITS-2 sequences (Woods et al., 2000; Gasser et al.,
2001; Lew et al., 2003; Haug et al., 2007) or sequence
characterized amplification regions (SCARs) (Fernandez
et al., 2003). Woods et al. (2000) described a polymerase
chain reaction-linked restriction fragment length polymor-
phism (PCR-RFLP) approach targeting the second internal
transcribed spacer (ITS-2) to characterize six Eimeria spp.
However, the ITS sequences show some variability both
within a genome as well as between species and strains
(Cantacessi et al., 2008).
3.5. Toxoplasmosis
Toxoplasma gondii is a tissue cysts forming coccidian
parasite that infects most warm-blooded animals; in utero
infection can result in foetal death in humans, sheep and
goats.
The demonstration of oocysts in cat faeces is possi-
ble by using standard flotation techniques, but definitive
diagnosis usually require sporulation of oocysts and fol-
lowed by bioassay in mice to distinguish them from other
closely related coccidian species (Dubey and Beattie, 1988).
However, cats only excrete oocysts for a short period after
primary infection and, therefore, various serological meth-
ods have been developed to detect humoral antibodies in
exposed cats and other animals. The CFT was the first sero-
logical tests to be developed to detect antibodies in exposed
individuals (Warren and Sabin, 1942); this was followed
by the Sabin–Feldman dye test (Sabin and Feldman, 1948),
which showed to be a very sensitive test. However, as live
tachyzoites are used in this procedure, which could poten-
tially pose a danger to the operator, other tests have been
developed. These include the immunofluorescent antibody
test, the direct agglutination test, the latex agglutination
test and the modified agglutination assay (Buxton, 1998).
Various ELISA methods using crude, fractioned or recom-
binant antigens have been developed (Dubey, 2009). The
modified agglutination test still appears to be the most
sensitive and specific of all the serological tests available.
Both animal inoculation and in vitro culture methods
have been used to demonstrate T. gondii in cases of abor-
tion. However, these techniques are slow and expensive
and rely upon submission of fresh material to the diagnos-
tic laboratory and, therefore, they are not routinely used
(Buxton, 1998). Immunohistochemical techniques, allow-
ing for the visualisation of T. gondii in tissue sections are
often used in the diagnosis of abortions (Buxton, 1998;
Dubey, 2009).
To overcome the limitations of the serological tests, var-
ious PCR, nested PCR and real-time PCR techniques have
been developed to detect T. gondii DNA in samples (Switaj
et al., 2005; Gutierreza et al., 2010). However, the PCR
diagnosis is not standardised and no consensus on the
primers/DNA targets to be amplified exists. Primers are
generally based either on the 18S rRNA-, P30-, B1-genes,
529 bp repeat fragment or the AF146527 element. PCR test
amplifying genes with high copy numbers in the genome
are more sensitive. Recently, Zhang et al. (2009) described
a LAMP method for the detection of T. gondii, by using the
529 bp repeat element of T. gondii, and it was found that
the assay was slightly more sensitive than the equivalent
PCR.
 T. de Waal / Veterinary Parasitology 189 (2012) 65–74
3.6. Neospora caninum infection
N. caninum is a tissue cysts forming coccidian parasite
closely related to T. gondii. It is recognised as a leading cause
of abortion in cattle and neurological disorders in dogs
(Dubey and Lindsay, 1996). It was first described in 1990
in a congenitally infected lamb in UK (Dubey et al., 1990).
However, it is generally not regarded as a significant cause
of abortion in sheep (Buxton, 1998). Since 2000 neosporosis
has been reported worldwide as cause of congenital infec-
tion in naturally exposed sheep and mortality in newborn
lambs. It recently has also been suspected in several cases
of abortion in New Zealand (Howe et al., 2012).
Various methods are used to confirm the involvement
of N. caninum in clinical problems of abortion or neu-
rological disorders. Immunohistochemical techniques are
commonly used to demonstrate parasites in the placenta
or foetal heart or brain tissue (Lindsay and Dubey, 1989).
The most common techniques for diagnosing N. can-
inum are those aimed at detecting specific antibodies in
sera; specifically, the immunofluorescent antibody test
(Conrad et al., 1993), the immunoblotting (Schares et al.,
1998), the direct agglutination tests (Packham et al., 1998)
and the ELISA have been used. Most ELISAs are based either
on whole tachyzoite lysate, fixed tachzoites or recom-
binant and have a diagnostic specificity and sensitivity
ranging between 78 and 99% (Lally et al., 1996; Von
Blumröder et al., 2004; Ortega-Mora et al., 2006). Sev-
eral competitive-inhibition (CI) ELISA have been developed
using parasite-specific monoclonal antibodies. A CI-ELISA
that detects serum antibody to a 65-kDa immunodom-
inant N. caninum tachyzoite surface antigen (p65) has
been developed and showed high sensitivity and specificity
(Ortega-Mora et al., 2006). The ELISA has also been adapted
to enable analysis of IgG avidity to distinguish between
acute and chronic infections (Bjorkman et al., 1999; Maley
et al., 2001). In dairy farms, individual or bulk tank milk
samples can be tested by ELISA and is a useful tool to iden-
tify those herds with high seroprevalence and to monitor
control programmes. An immunochromatographic test has
also been developed for the detection of N. caninum anti-
bodies (Liao et al., 2005).
PCR techniques, based on the amplification of the Nc5
repeat sequence or the ITS-1 region have been useful as
diagnostic tools for detection of the parasite in aborted
foetuses (Holmdahl and Mattsson, 1996; Muller et al.,
1996). To increase the sensitivity of semi-nested or nested
PCR, various modifications have been suggested, although
there does not appear to be a clear relationship between
the PCR format and the sensitivity (Van Maanen et al.,
2004). A quantitative real time PCR has been developed
using SYBR Green 1 (Collantes-Fernandez et al., 2002).
A new method of using indirect in situ PCR has been
described, whereby the amplified products were detected
using a primed in situ (PRINS) reaction with hapten-labeled
nucleotides and visualised using fluorochrome-labeled
antibodies (Loschenberger et al., 2004).
4. Concluding remarks
Serological tests are often applied in the international
trade to prevent the introduction of protozoan parasites
71
into areas or farms, where the respective diseases do not
occur. The development of molecular tools has allowed the
diagnosis, as well as the study of the genetic variability of
pathogens and the identification of species-specific mark-
ers. Frequently, the bottleneck/difficulty for an effective
molecular diagnostic procedure is not the PCR amplifica-
tion of the genomic DNA, but rather the preparation of the
DNA. The environmental stages of many of the protozoa
have thick walls that are resistant to chemical and mechan-
ical forces; for example, effective techniques are needed to
rupture the oocysts wall of coccidian species before DNA
extraction. The inhibitors present in faecal material also
have negative effect on sensitivity, because of the loss of
material due to the purification steps needed. Major chal-
lenge remains to develop specific, fast and cheap diagnostic
test for the identification of protozoan parasites in animals
that could still be applied to remote areas with limited
infrastructure.
Conflict of interest statement
The author declares that there is no conflict of interest.
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