VETERINARIA 2015 | Volume 2 | Issue 1 | Pages 1-6

Review article
Recent Diagnostic and control approaches in Equine piroplasmosis
Deepak Sumbria and LD Singla*
Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-
141004, India

Abstract
Equine piroplasmosis (EP) is a tick-borne disease of horses, mules, donkeys and zebra caused by the intra erytrocytic
apicomplexan haemoprotozoan parasites Babesia caballi and Theileria equi. The introduction of carrier equines into areas in
which tick vectors are present can lead to an epizootic spread of the disease. International movement of equines broaches a
serious threat due to equine piroplasmosis. The EP is caused by, Babesia caballi and Theileria equi. Earlier, both conventional
microscopic and serological test [complement fixation test (CFT), indirect fluorescent antibody test (IFAT), and enzyme linked
immunosorbent assay (ELISA)] dwindle to diagnose the equines having the latent infection. Now to detect the parasites, more
specific, sensitive and accurate PCR based techniques for molecular diagnosis are available. Up till now, no vaccine has been
augmented, thus accentuating the need for accurate diagnosis and treatment of this haemoparasite. For meticulous diagnosis of
EP, the test to be considered as a "gold standard" if it is sensitive enough to detect early both acute and chronic infections,
specific for the differentiation between the two parasitic species, and it should be economical. For complete elimination of the
EP, disease control programs must be integrated with the application of acaricides and grazing management policies. Moreover,
for long-term prevention and control, it is important to have one or more vaccines directed against blood-feeding ticks and/or
against B. caballi and T. equi sporozoites, or other stages of the hemoprotozoans that can be used to elicit a protective immune
response in the host.
Keywords: Equine piroplasmosis, Babesia caballi, Theleria equi, Diagnosis, Treatment, Control.
Received December 10, 2014; Revised February 1, 2015; Accepted February 4, 2015
*Corresponding author Dr. L.D. Singla Email: ldsingla@gmail.com Phone: +91-9316061974

To cite this manuscript: Sumbria D, Singla LD. Recent diagnostic and control approaches in equine piroplasmosis. Veterinaria
2015;2(1):29-32.

Introduction node biopsies along with the clinical signs

The total equine population in the world is about 59
million horses, 44 million donkeys and 11 million
mules [1]. In most of the developing countries, more
than 72% of the worlds horse population and over
97% of the worlds donkey and mule populations are
kept specifically for work. Among the multiple health
and welfare problems affecting working equids,
parasitic diseases such as equine piroplasmosis (EP)
are one of the major constraints of their and
performance and productivity, often leading to high
morbidity and mortality.
The EP was first reported in Sudan by Oliver
(1907) cited in Abdoon [2]. It is a disease of equids
caused by the intracellular, haemoprotozoan parasite
Babesia equi, reclassified as Theileria (T) equi, [3]
and Babesia (B) caballi. In 1901 Theiler [4], perceive
intra-erythrocytic parasites in blood samples. The
clinical signs of the haemoparasitic infection
observed in equids in Pretoria, South Africa, were
similar to human malaria infection (plasmodiidae) so
he assigns it to be equine malaria. Later on Laveran
[5] acclaims it as intra erythrocytic piroplasms. Blood
and /or lymph node fluid smears microscopic
detection of the piroplasms and schizonts from the
suspected host is the true Gold Standard diagnostic
test available for EP still, in T. equi infection lymph
1
(enlargement of lymph nodes and high fever of
104F) of the disease also help in detection. These
tests are brawny, but are totally dependable upon the
qualified laboratory technicians and have poor
susceptivity. Parasitological identification of the EP
is usually hampered in the case of carrier animals and
long standing chronic cases due to lower graded
parasitemia. To vanquish the shortfalls of slide
examination, more advanced serological tests have
been endeavor. These tests perceive circulating
antibodies by using either piroplasms or cultured
macro schizonts as the antigen.
These serological tests do not distinguish
between present and past infection and residual
antibodies from previous vaccination programs.
Identification of carrier animals is important for the
evaluation of infection risk as they serve as a pool of
infection for ticks and cause natural transmission of
the disease. For the avant-garde identification, both
sensitive and specific tests are required. Recently, in
vitro culture from blood was used diagnostically for
the identification of carrier animals with B. equi [6].
Direct detection of T. equi and B. caballi nucleic acid
from equines blood with DNA probes detected a
higher number of carrier animals than microscopic
examination methods. With the aim of developing
more rapid and sensitive diagnostic alternatives,
VETERINARIA
polymerase chain reaction (PCR) based systems were
designed to detect the genomic DNA of T. equi and
B. caballi. So, for effective detection of EP both
serological and molecular techniques should be used.
Here various diagnostic methods which can be used
in combination for proper and timely diagnosis of EP
as this disease posed a great threat to intercontinental
movement of equids across the world are discussed.
1. Blood smear identification of the EP
Best diagnosis depends on the identification of the
parasite in thin or thick blood smears stained with
Giemsa using light microscopes [7]. Direct
parasitological verification of chronic B. caballi
infection is almost unfeasible, but is occasionally
successful with T. equi. In general, chronic or in-
apparent infections can only be confirmed after
transfusion of approximately 500 ml of blood in
susceptible animals. The parasites are best
demonstrated by staining blood smears with 10%
Giemsa solution [8]. However, the accuracy of
microscopic examination depends upon the skill and
experience of the scientist investigating the smear,
moreover it is very difficult to demonstrate the
parasite from latent parasite carrier animal because of
low parastaemia (<0.01-0.001%, implies one infected
RBC out of 104-105 RBC). The carrier state in EP
commonly presents a special problem of diagnosis
since no outward signs of the disease are evident [9].
Diagnosis of these parasites is mainly based on
conventional romanowsky stains, which are having
their own disadvantage of missing the low
parasitemic sample and some time confusion with
staining particle. But, Acridine Orange staining
enables more rapid detection and identification of
blood parasite. As it is a fluorochrome stain, giving
red fluorescence of ribonucleic acid (RNA) and a
green fluorescence of deoxy ribonucleic acid (DNA).
Thus, this method offers higher sensitivity compared
to romanowsky staining which can detect one
parasite par 107 erythrocytes compared to the
detection of one parasite per 104 to 105 erythrocytes.
Moreover, it is more accurate because it stains
nucleus/DNA of the parasite which excludes the
confusion caused by staining debris in conventional
staining. The technique enables more rapid detection
and identification of blood parasites for the detection
of blood parasites.
In sub-acute and chronic infections it is often
difficult to detect the parasites in thin smears due to
the low level of parasitaemia; however, examination
of thick blood smears may be a useful addition. Now
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2015 | Volume 2 | Issue 1 | Pages 1-6
a day, in vitro culture method has become a valuable
tool. This technique is found to be sensitive is able to
successfully confirm the carrier animals, but is
cumbersome and time consuming.
2. Biological tests
Isotest
Measured quantities of washed red cells or whole
blood (500 ml) are transfused into a susceptible
splenectomised equine. This animal is then kept
under close observation for clinical signs of disease.
After observation of any alteration, the blood samples
can be taken from the equines. The diagnosis is
confirmed by the presence of protozoans in the red
cells by microscopical examination under oil
immersion lens. But, this test proves to be a
cumbersome and expensive exercise.
Xenotest
In this technique the parasite free tick vectors are
allowed to feed on the suspected equines. Then,
either these ticks are examined for the presence of
parasites or ticks are further allowed to feed on
susceptible healthy equines, leading to further
transmission of the disease. The blood samples of
these equines are further taken for the final diagnosis
of the parasites in erythrocytes by microscopical
examination.
3. In vitro culture technique
For cultivation of Babesia bovis, suspension culture
was developed, which was later modified to spinner
flask culture so that continuous growth of the parasite
can be obtained [10]. Spinner culture showed slow
growth rate of parasite and require tedious daily
maintenance so MASP (micro aerophilous stationary
phase culture medium) was developed. In this
medium defibrinated infected blood is suspended to
final PCV of 5-10% in a medium containing 40%
foetal bovine serum. In MASP there is a low oxygen
atmosphere which is obtained by a column of
medium above the parasitized cell and its act as a
barrier to oxygen exchange, so preventing its side
effects. This culture was developed for maintaining
Babesia bovis and B. bigemina [11]. The babesia can
be maintained up to three months in MASP. Later
this culture was modified for continuous culturing of
B. equi and B. caballi. B. equi requires a humid gas
mixture of 2% O2, 5% CO2 and 93% N2 as compared
with humidified 5% CO2-in-air. Zweygar et al [12]
further made 2 culturing media (SFRE-SFRE and
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HL-HL medium), with which B. equi can be cultured
at normal oxygen tension in the incubator. If
compared with blood films, culture is expensive and
requires a skilled personal and thus cant be used in
laboratory procedure, moreover for conformation 2-
10 days are required in culturing.
4. Serological tests
Complement fixation test (CFT)
The CFT was developed in 1945 [13]. In the past, in
some countries CF test has been used to qualify
horses for importation [14]. It relies on fixation of
complement during a reaction between the specific
antigen and antibody [15, 16]. Fixation of the
complement can be assessed by estimation of lysis as
a percentage, i.e. 0%, 25%, 50%, 75%, and 100%,
which are accorded values of 4+ positive, 3+
positive, 2+ positive, 1+ suspicious trace (negative)
and complete haemolysis (negative). Horses with sera
which react positively at a dilution of 1:5 (i.e. less
than 25% lysis) are considered positive. The CFT
detects antibodies as early as 8 days after infection;
whereas, titers decline in 2 to 3 months after
exposure. It is a specific test for acute infections, but
has low sensitivity in chronic infection. The CFT was
first implemented as an import test for equids
entering the United States in 1970 and remained the
official test until 2005. The CF test may not identify
all infected animals, especially those that have
produced anti-complementary reactions like donkey
and zebra, because of the inability of IgG to fix
guinea-pig complement [17]. Therefore, the IFA test
and C-ELISA have replaced the CF as the prescribed
tests for international trade.
Indirect immunofluorescent antibody test (IFAT)
IFAT was first used for the sero-diagnosis of B.
caballi in year 1964 [18]. The IFA test can be
successfully used to perform a differential diagnosis
of T. equi and B. caballi infections [19]. It is a more
sensitive test than the CFT. In this parasite, antigens
are bound to a glass slide and allowed to react with
test sera. Antibodies that bind with slide are visible
under ultraviolet light after binding of the
fluorescein-labeled anti equine sera. If sera, show
strong fluorescence of the parasites at a dilution of
1:80 or higher it is considered positive. To increase
specificity with the IFAT, the serum must be diluted,
resulting in a concurrent loss of sensitivity [15].
The recognition of a strong positive reaction is
simple, but any differentiation between weak positive
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2015 | Volume 2 | Issue 1 | Pages 1-6
and negative reactions requires considerable
experience in interpretation. On the other hand IFAT
is time consuming, requires large amounts of antigen
and because of the subjectivity in interpreting
fluorescence, it is difficult to standardize. The IFAT
is used as an adjunct test to aid in analysis of CFT
results, but it remains one of the prescribed tests for
equine piroplasmosis recommended by the OIE.
Enzyme-linked immunosorbent assay (ELISA)
The ELISA is used to detect antibodies for both
species of the parasite in experimentally infected
horses. It is apparent that cross-reactions between T.
equi and B. caballi occur. Competitive ELISA (C-
ELISA) overcomes the problem of antigen purity
because the specificity of the test depends only on the
monoclonal antibody used. For the detection of T.
equi infection, Knowles et al [20], used T. equi
equine merozoite antigen (EMA)-1 and specific
monoclonal antibodies, and developed a competitive
inhibition C-ELISA. This C-ELISA was later
improved by using recombinant protein instead of
culture-derived whole parasites [21]. Kappmeyer et
al [22] developed C-ELISA using recombinant B.
caballi rhoptry-associated protein-1 (RAP-1). In a
field survey, this test identified 25 percent more sera
positive for B. caballi than CFT. In 2004, the OIE
approved the C-ELISAfor both T. equi and B.
caballi detectionas a prescribed test for
international horse movement [23]. Knowles et al
[20, 24] had developed a monoclonal antibody
(mAb)-based competitive ELISA (cELISA). A mAb
(36/133.97) was selected from a panel of mAbs,
which reacted with a 34 kDa surface protein of T.
equi also detected by immune sera of horses infected
with different strains of the parasite. In this test,
antibodies in test sera compete with the mAb for the
antigenic site in the merozoites. Positive titers are
detected by a reduced color reaction. Babesial
antigens between the ranges of 141-30 kDa were
demonstrated to be dominant, among these proteins
of molecular weight (MW) 141, 112, 70, 50, and 48
kDa were recognized by the immune sera tested. In
B. caballi infections the proteins of MW 50 and 48
kDa were considered suitable for use in an
immunodiagnostic test, since in Western blots a wide
range of B. caballi sera had recognized these antigens
[25].
Now a day, number of recombinant antigens for
the use in ELISAs such as Recombinant T. equi
(EMA-1; EMA-2; Be82 and Be158) and B. caballi
proteins (RAP-1; Bc48; Bc134) are produced in
VETERINARIA 2015 | Volume 2 | Issue 1 | Pages 1-6
Escherichia coli or in insect cells by baculovirus. A 6. Treatment
competitive inhibition ELISA (C-ELISA) using
EMA-1 protein and a specific monoclonal antibody The main aim of treatment is to eliminate of
(MAb) that defines this merozoite surface protein haemoprotozoan parasites from equines. There are
epitope have been used in a C-ELISA for T. equi a number of drugs available for the treatment of
[18]. EP. T. equi has been reported to be more refractory
to babesiacidal drugs than B. caballi. In old days,
5. Molecular techniques Bisazo dyes such as Trypan blue were used against
B. caballi but not for T. equi. This drug causes the
Overall, the sensitivity of the CFT to detect T. equi
discoloration of the animal's tissues. Other drugs
came out to be 47% and the C-ELISA was 96%
which are effective in eliminating EP infections
after complete statistical analysis [22, 24, 26]. The
include diminazene diaceturate, imidocarb and
specificities of the 2 tests were found to be 94%
amicarbalide. Antitheilerial compounds, including
and 95%, respectively. For testing of B. caballi,
parvaquone and buparvaquone, have been shown to
the sensitivity of the CFT is 88% and the C-ELISA
reduce parasitemia of initial infection, but failed to
is 91%. The specificities of the 2 tests for B.
sterilize T.equi infections completely. Diminazene
caballi are 98% and 70%, respectively. Polymerase
diaceturate causes swelling and necrosis at the
chain reaction detects the presence of the organism
injection sites. Depression, respiratory distress, and
of interest by amplifying and detecting specific
other signs of intoxication are often the result of
fractions of the DNA. This test is sensitive and has
toxic doses and thus careful monitoring is needed.
been utilized in research settings for the detection
For the treatment of babesiosis, imidocarb has been
of the EP. Many variations of PCR are there, but
used for more than 20 years but minimal
some important are-conventional or primary PCR
information was available about the
(one set of primers); real-time PCR (quantifies the
pharmacokinetic behavior of the drug in equids. In
level of parasite in peripheral blood); nested PCR
2002, Belloli et al [33] study showed that: (1) for
(two sets of primers used to increase sensitivity);
complete elimination of the drug from tissues, a
and nested PCR with hybridization (probe specific
prolonged period was required; (2) this drug was
for gene target results in enhanced sensitivity and
effectively distributed to tissues and (3) there was
specificity). Nested PCR for T. equi using the
evidence of possible sequestration of imidocarb in
EMA-1 gene sequence has been shown to detect a
vascular and extra vascular compartment. The liver
positive result equivalent to a percent parasitemia
was reported to be a storage tissue for imidocarb.
of 0.000006% [27]. One report also indicated that
Main adverse effects of Imidocarb are dose-
nested PCR detects 3.69% more infections that
dependent hepatotoxicity and nephrotoxicity [34].
microscopy and 2.29% more than traditional PCR
Increasing levels of Imidocarb were associated
[28]. For primary and nested PCR Babesia 18S
with increasing mortality and morbidity (local and
rRNA was widely used by various workers [28]. In
systemic reactions). Mortalities were attributed to
2007 LAMP [Loop Mediated Isothermal
acute renal cortical tubular necrosis and acute
amplification] targeting EMA-2 for T. equi and
periportal hepatic necrosis induced by two
Bc48 for the B. caballi detection had been used by
injections of 16 or 32 mg/kg of imidocarb [35].
Alhassan [29] and his coworker.
Some drugs may also be used in both B. caballi
Real time assays exploits the 5' nuclease
and T. equi infections. Acridine dyes, e.g.
activity of Taq DNA polymerase cleaving a dual
euflavine and others in this group can be
labeled fluorescent probe which has annealed to a
administered intravenously at a recommended dose
specific sequence between the two primers [30].
of 4-8 ml 100 kg-1 of a 5% solution with a
To date, the applications of real time PCR for the
maximum inoculums of 20 ml.In vitro growth
detection of tick borne disease pathogens have
inhibition of T. equi and B. caballi was reportedly
been described for Theileria sergenti and
induced by exposure to triclosan, with no adverse
Anaplasma phagocytophilum [31]. Real time PCR
effects on host cells [36]. In Supportive care would
has wider acceptance over other PCR techniques
involve management of anemia through blood
due to its improved rapidity, sensitivity,
transfusion and fluid treatment in animals with
reproducibility and the reduced risk of carryover
pigmenturia or dehydration.
contamination [32].

4
VETERINARIA
7. Control protocols
Complete Examination of horses for ticks
For tick examination the following protocol should be
strictly followed:
Always beginning at the horses head, examines the
both ears and palpate inside of each ear, examine the
false nostrils visually and with the forefinger palpate it.
Now move to the forelock and examine it, continuing
down the mane to the withers. Examine other area like
the sub mandibular/inter mandibular space, the axilla,
posterior fetlock to the coronet of the feet,
udder/scrotum area on both sides, tail and perineum
region. Always perform hand hygiene after examining
each animal, if any infected animal is found, then isolate
it from other healthy flock.
Removing attached ticks
Many methods are used to remove attached ticks from a
host. Most common methods include applying 70
percent isopropyl alcohol, fingernail polish, petroleum
jelly, or a hot kitchen match to the attached tick [37].
These procedures increase the risk of secondary
infection around the bite location. The best method for
tick removal was described by Needham [37]. Another
most common method for removing ticks is spraying
the ticks with pyrethrins or pyrethroids containing
aerosol repellent, and then spraying again within1
minute. Ticks will fall off after treatment.
Preventing tick infestations
Most important step in EP control program is to prevent
equine tick exposure, but it seems to be complicated
because of specific biological requirements of a tick
species and whether a ticks life cycle is dependent on
one host or multiple hosts. If the tick requires multiple
hosts for its life cycle, then access to favorable questing
sites, host availability, and seasonal changes in tick
increases and it in turn play a vital role in transferring
EP. One host ticks are easy to control.
Control of ticks by chemical compound
Chemical control methods are effective in reducing host
exposure to ticks within a short span of time. The most
common compounds used are natural pyrethrins
(derived from seed cases of the Chrysanthemum
cinerariaefolium plant and consisting of two stereo
isomers, pyrethrin I and pyrethrin II, along with a
cyclopropane core); synthetic pyrethrins (often called
pyrethroids) and carbamate insecticides (e.g., Sevin
with the carbaryl, 1-naphthyl methylcarbamate, as the
active ingredient). Due to the insecticidal property of
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2015 | Volume 2 | Issue 1 | Pages 1-6
natural compounds, pyrethrins act as potent neurotoxins
for insects moreover, in low or nonlethal doses,
pyrethrins had repellent properties [38]. In cattle the
most widely approved acaricide is amitraz (a
triazapentadiene compound) which cannot be used on
horses because it can cause irreversible gut stasis. For
equines the most common acaricide formulations are
natural and synthetic pyrethrins. Often these
formulations include piperonyl but oxide as a synergist
that inhibits rapid metabolism of the active compound
by arthropods, thus prolonging the neurotoxic effects.
Other synergists that may be added are: chloropyriphos,
dichlorvos, or thiazolyn [39]. For effective tick control,
in equine the frequently used Pryrethroid formulations
are: cypermethrin, alphamethrin, tetramethrin,
prallethrin, and deltamethrin.
Now a days due to indiscriminate use of chemical
compound for tick control, the problems of acaricide
resistance in ticks, animal product contamination, and
environmental residues had arisen. These compounds
are toxic to many aquatic organisms, waterfowl, and
bees [40]. In case of human toxicity to pyrethrins is
considered low, fatal asthma has been reported after the
use of an animal shampoo containing pryethrin [41].
Biological/nonchemical control of ticks
As nowadays tick is becoming resistant to various
chemical compounds so an alternate method to control
the ticks population should be used which are more eco
friendly and safe. In addition to habitat and pasture
management, other nonchemical methods of tick control
emphasis on limiting movements of free-ranging
wildlife hosts, such as deer. It includes deer repellents,
deer fencing, and substituting less palatable plants on
pasture. In South Africa ticks repellent plants (leaves of
Aloe ferox, the bark of Ptaeroxylon obliquum) have
been tried by Moyo and Masika [42].
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