Review

J Vet Intern Med 2013;27:1334–1346

Review of Equine Piroplasmosis

L.N. Wise, L.S. Kappmeyer, R.H. Mealey, and D.P. Knowles
Equine piroplasmosis is caused by one of 2 erythrocytic parasites Babesia caballi or Theileria equi. Although the genus
of the latter remains controversial, the most recent designation, Theileria, is utilized in this review. Shared pathogenesis
includes tick-borne transmission and erythrolysis leading to anemia as the primary clinical outcome. Although both para-
sites are able to persist indefinitely in their equid hosts, thus far, only B. caballi transmits across tick generations. Patho-
genesis further diverges after transmission to equids in that B. caballi immediately infects erythrocytes, whereas T. equi
infects peripheral blood mononuclear cells. The recent re-emergence of T. equi in the United States has increased
awareness of these tick-borne pathogens, especially in terms of diagnosis and control. This review focuses in part on fac-
tors leading to the re-emergence of infection and disease of these globally important pathogens.
Key words: Anemia; Babesia caballi; Erythrocytic parasite; Theileria equi; Tick-transmitted.

quine piroplasmosis is an infectious, tick-borne
E disease caused by the hemoprotozoan parasites
Theileria equi and Babesia caballi. Piroplasmosis,
which is also known in the literature as equine babesi-
osis, theileriosis (concerning T. equi), and biliary fever,
affects all equid species, including horses, donkeys,
mules, and zebras.1,2 Infection with either or both of
these obligate, intraerythrocytic organisms can cause
varying degrees of hemolytic anemia and associated
systemic illness. As T. equi infection “silently” re-
emerged in the United States, questions are being
raised concerning the tick-vector-parasite-host require-
ments necessary for the development of clinical dis-
ease. The parasites and their natural tick vectors are
endemic to most countries with tropical and subtropical
climates.3–5 The goals of control and disease eradication
vary tremendously between endemic and non-endemic
nations. Recent outbreaks of infection with limited dis-
ease expression in the United States and the Netherlands
coupled with the identification of novel vectors within
the United States have prompted a renewed interest in
this historically important disease.6–9
Taxonomy of the causative agents of piroplasmosis
has been in question since their discovery and
remains controversial for T. equi.10,11 Currently, the
parasites are classified within the phylum Apicom-
plexa, which contains other hemoprotozoan such as
Plasmodium and Theileria. The parasite, termed Piro-
plasma equi (reclassified later as B. equi), was first
recognized in South Africa as the causative agent of
disease in 1901.12 A few years later, it was discovered
From the Department of Veterinary Microbiology and Pathology,
Washington State University (Wise, Mealey, Knowles); the Animal
Disease Research Unit, U.S. Department of Agriculture,
Agricultural Research Service (Wise, Kappmeyer, Knowles), and
the OIE Reference Laboratory for Equine Piroplasmosis (Knowles),
Pullman, WA.
Corresponding author: L.N. Wise, DVM, DACVIM, Depart-
ment of Veterinary Microbiology and Pathology, Washington State
University, PO Box 646630, Pullman, WA 99164; e-mail:
nwise@vetmed.wsu.edu.
Submitted February 5, 2013; Revised April 15, 2013;
Accepted July 16, 2013.
Published 2013. This article is a U.S. Government work and is in
the public domain in the U.S.A.
10.1111/jvim.12168
that 2 separate and distinct parasites could infect
equid erythrocytes, one significantly larger than the
other.13 The larger of the parasites was termed
Piroplasma caballi, only to be later reclassified as
B. caballi.14 Whereas B. caballi is considered a classic
“babesia” species, the taxonomy of B. equi remains
controversial. Based in part on finding an extra-eryth-
rocytic stage within equine PBMC, B. equi was reclas-
sified as T. equi in 1998.11 Molecular phylogenetic
investigations indicated that the organism possesses
characteristics of both babesia and theileria lineages,
possibly placing it between the two.15–18 Recent geno-
mic analysis supports the concept of a new genus for
T. (B.) equi.10 Additional data are needed to deter-
mine the final placement of this parasite and there-
fore this review will use the most recent designation,
T. equi.
Epidemiology/Ecology
Piroplasmosis occurs in most countries worldwide
and infection is maintained within equine populations
as long as competent vectors are present.19 For
T. equi, the reservoir is the persistently infected equid;
however, for B. caballi, both infected horses and the
primary tick vector are reservoirs.20–24 Although the
precise tick-vector-parasite-host requirements for infec-
tion or clinical disease are not known, the outcome of
increasing densities of infected horses and ticks in an
area is an increase in infection and potentially disease.25
Clinically silent transmission appears common.7 The
risk of life-threatening clinical disease increases with
the presence of factors such as immunological naivety
and increased density of infected ticks and horses.26
Although numerous studies have been published
regarding the epidemiology and distribution of infec-
tion within specific countries and regions, these
publications should be interpreted with caution given
the profound variation in experimental design, sample
population, and diagnostic testing. Particularly impor-
tant is the previous use of the complement fixation test
(CFT) for detection of antibody against B. caballi
or T. equi. Abundant data have shown that the CFT
lacks sensitivity in detecting persistent infections.27
Ixodid tick vectors occur in tropical, subtropical,
and some temperate climates.28 T. equi is more preva-
 Equine Piroplasmosis
lent than B. caballi, yet B. caballi has been identified
in more northern regions of the Northern Hemisphere
than T. equi.1 Given the currently available informa-
tion on geographic distribution of infected horses
according to the Office International des Epizooties/
The World Organisation for Animal Health (OIE),
Central and South America, Cuba, Africa, Asia, the
Middle East and Southern Europe are considered
endemic regions.8 Within South America, the disease is
readily identified in all regions with the exception of
the southernmost areas of Chile and Argentina. The
prevalence and distribution of the infection within the
Caribbean nations are questionable, but the disease
has been reported on most islands, including Trinidad
and Cuba.8,29,30 Disease is widespread in Africa and
Asia with the highest prevalence reported in South
Africa.31,32 Not all countries report identified cases to
the OIE, making an accurate understanding of the cur-
rent parasite distribution difficult. Countries such as
Mexico and China are not considered endemic because
the OIE routinely receives no information regarding
distribution of piroplasmosis cases in those countries.
Yet, articles from both Mexico and China have been
published citing cases within those countries.33–35
Compiling a list of currently non-endemic regions is
equally challenging given the difference in surveillance,
import/export restrictions, and disease reporting that
occurs. Current disease status for those countries that
report to the OIE can be found on their website.36
Detection of infection within the United States in
recent years has placed the “piroplasmosis free” status
of this country under scrutiny.7,37 The 1st case of B. cab-
alli was identified in a horse in 1961 in southern Florida
and while the source of infection was never definitively
determined, it was speculated that it was attributable to
importation of horses from Cuba.38,39 During the fol-
lowing decade during extensive surveillance programs,
several hundred total cases of both B. caballi and
T. equi were diagnosed in 7 states. Dermacentor (Ano-
center) nitens, a tick capable of transmitting B. caballi,
was identified in affected Florida counties, yet this docu-
mented vector’s role in disease transmission was never
confirmed. All infected horses were deported, quaran-
tined, or treated with various drugs until a negative
serologic result was obtained. Intense surveillance of
horses and ticks continued in Florida until 1988 when
the United States was deemed free of disease. To
maintain this status, USDA APHIS improved restric-
tions on importation of horses from endemic areas.
In 2008, 20 T. equi-infected horses were identified
on 7 separate premises in Florida.37 All affected horses
were associated with horses that had been imported
from Mexico and all were engaged in illegal horse rac-
ing. Given the history and distribution of infected
horses, inappropriate management practices including
needle sharing and “blood doping” were assumed to
be the mode of transmission. No tick vectors were
identified despite aggressive surveillance. More recently
in 2009, an outbreak of T. equi was identified on a
ranch in southern Texas involving approximately 400
horses.7 All infected horses resided on the premises or
1335
had been at some time associated with the ranch. An
investigation of this property allowed identification of
2 previously unrecognized competent vectors of T. equi
within the United States (Amblyomma cajennense
and Dermacentor variabilis). Ticks collected from the
infected horses on the premises were capable of biolog-
ically transmitting disease to na€ıve horses.9
Competent ticks and iatrogenic blood transfers are
efficient modes of transmission.19–21,25,40 With over 850
tick species worldwide and approximately 85 within
the United States, the potential for transmission is
high.28 However, the presence of a competent tick vec-
tor and infected horses within the same area does not
always lead to further infection or disease. Many fac-
tors must be considered including season, climate,
host-specificity, and the particulars of a competent
tick’s life cycle.25
The life cycle of a tick involves 4 life stages: egg,
larva, nymph, and adult. After hatching from an egg,
the larva feeds on its host and molts into a nymph.
The nymph then feeds and molts into an adult.
Females and males proceed through these life stages,
but the female dies after laying her eggs. Adult male
ticks seeking females have the potential to feed on
multiple hosts. Ticks are classified as hard ticks (Ixodi-
dae) or soft ticks (Argasidae).28 Both are vectors for
pathogen transmission, yet only hard ticks are natural
vectors for B. caballi and T. equi.
Tick transmission can occur via 3 forms: intrasta-
dial, transtadial, or transovarial. Intrastadial transmis-
sion occurs when acquisition and transmission of the
parasite occurs within 1 life stage (no stage transition
before transmission). Transtadial describes acquisition
of infection in 1 stage and the ability for the same tick
to transmit the infection during subsequent life stages.
The parasite is maintained within the tick as it devel-
ops. Transovarial transmission occurs when the female
acquires parasites, which enter ovaries and are trans-
mitted to offspring, allowing maintenance of the para-
sites across tick generations.20
Multiple ixodid tick species have been identified as
either natural or experimental vectors of piroplasmosis.
B caballi is transmitted by 15 separate species (7 Der-
macentor [Anocenter] sp., 6 Hyalomma sp., and 2
Rhiphicephalus sp.) and T. equi by 14 species (4
Dermacentor sp., 4 Hyalomma sp., 5 Rhiphicephalus
(Boophilus) sp., and A. cajennense). B. caballi is trans-
mitted transtadially and transovarially by its vectors.41
T. equi is generally transmitted through transstadial
and intrastadial transmission.20 Transovarial transmis-
sion of T. equi occurs, but the precise role in epidemi-
ology has not been detailed.42,43
Discovery of the vector D. reticulatus in the Nether-
lands in 2010 combined with recognition of a subclini-
cally B. caballi-infected horse led to a surveillance of
that area that resulted in identification of several of
T. equi- and B. caballi-infected horses.6 Before 2009,
only 2 tick species known to transmit T. equi naturally
had been identified within the southernmost parts of
the United States: D. nitens and R. microplus. Within
the Texas outbreak, infected horses were found to be
1336
infested with 4 different tick species: A. cajennense,
A. maculatum, Dermacentor (Anocenter) nitens, and
D. variabilis. A. cajennense was the most abundant,
being identified on approximately 79% of the infected
horses, followed by A. maculatum (19%), D. variabilis
(16%), and D. nitens (3%). Before this outbreak,
A. cajennense had not been identified as a vector for
T. equi. The geographic distribution of these 3 host
ticks within the United States appears to be limited to
Texas and Florida.44 The role of A. maculatum in the
outbreak currently remains unclear and although
D. nitens is a known vector of B. caballi, evidence
supporting transmission of T. equi is lacking. Adult
D. variabilis ticks collected from horses on the ranch
were able to experimentally transmit disease to na€ıve
horses, but their role as a natural vector in this
outbreak remains questionable.9
Pathogenesis (Transmission/Life Cycle of
Parasites)
The life cycle of both B. caballi and T. equi involves
distinct stages that occur in the host and tick45,46
(Fig 1, 2). Both parasites progress through 3 life
stages: the sporozoite (asexual transmission stage), the
merozoite (asexual blood stage), and the gametocyte
(sexual blood stage). The development of these para-
sites within the tick can vary depending on species of
tick involved. Regardless of species variation, for both
B. caballi and T. equi, infectious sporozoites are trans-
mitted through the tick-saliva to the equid host. The
duration of the extrinsic incubation period (acquisition
feed; parasite replication time in the tick vector and
transmission feed) is not defined for B. caballi or
T. equi. Once within the host, B. caballi sporozoites
directly invade erythrocytes where they multiply and
develop first into trophozoites and then into merozo-
ites. After erythrocyte rupture, merozoites are released
Fig 1. Life cycle of Babesia caballi. Illustration by Massaro Ueti.
Wise et al
and invade other erythrocytes. T. equi’s initial invasion
is different in that it first enters PBMCs.11 This life
cycle event is part of the justification for the most
recent taxonomic classification as Theileria.10 Inside
PBMCs, T. equi sporozoites develop into large schizo-
nts and after approximately 9 days, merozoites are
released and invade erythrocytes. For both parasites,
asexual replication results in an expanding population
of merozoites and parasitized erythrocytes. Some mer-
ozoites develop into gametocyte forms within equine
peripheral blood. Upon ingestion of merozoites (and/
or gametocytes) by a competent tick, the parasites
undergo sexual reproduction, with gametocytes devel-
oping into gametes, which combine to form zygotes
within the tick midgut. The zygotes develop differently
depending on the tick species and the parasite. After a
period of 6–24 days, continued development results in
the presence of sporozoites within the salivary gland of
the tick.43,47,48
Transmission can occur iatrogenically through inap-
propriate mixing of the infected and uninfected
blood.37 This occurs most frequently during the prac-
tice of needle sharing between positive and na€ıve
horses, but use of any blood-contaminated equipment
could result in transmission.49 Infection can also be
caused when chronically infected horses serve as blood
donors to na€ıve horses. The illegal practice of “blood
doping” (prerace blood transfusions) was implicated in
the 2008 Florida outbreak.37 Experimental infection
can be achieved through intravenous and subcutaneous
routes as well as tick transmission.20,50–52
Although some details of pathogenesis remain
unknown, infection with either T. equi or B. caballi
causes erythrocyte lysis resulting in varying degrees of
hemolytic anemia. Physical rupture of erythrocytes
during release of merozoite stages causes intravascular
hemolytic anemia. Infected red blood cells are removed
from the circulation by splenic macrophages further
 Equine Piroplasmosis
Fig 2. Life cycle of Theileria equi. Illustration by Massaro Ueti.
contributing to hemolytic anemia. Nonparasitized
erythrocytes are also removed from circulation, but
the reason for this phenomenon is unknown. Data
from experimentally infected splenectomized donkeys
indicate that the biochemical structure of erythrocyte
membranes changes dramatically during infection with
T. equi.53 It was suggested that this conformational
change causes decreased deformability of the red cells,
which could lead to reduced microvascular blood flow.
Plasma levels of malondialdehyde (marker of lipid per-
oxidation) are significantly increased, suggesting that
an accumulation of oxidative ions also contributes to
erythrocyte lysis.53 These parasites also alter coagula-
tion in infected horses through unknown mechanisms.
B. caballi-infected erythrocytes cause microthrombi by
clumping within small vessels, leading to venous stasis
and vasculitis.52,54 Varying degrees of thrombocytope-
nia and prolonged clotting times have been reported
during infection with T. equi and B. caballi.55 Hypoth-
eses regarding the pathogenesis of decreased platelet
counts include immune-mediated destruction, splenic
sequestration and/or excess consumption as is
observed in disseminated intravascular coagulation.
Severe piroplasmosis can result in hypercoagulability,
systemic inflammatory response syndrome, and subse-
quent multiorgan system dysfunction.56
Placental transmission from infected carrier mares to
their fetuses has been documented.15,57–59 This trans-
mission can result in abortion (most commonly in late
gestation), stillbirth, or neonatal infection and can
occur across placentas that are histologically normal.
The prevalence of this type of transmission is
unknown. The natural outbreak documented in Texas
of 2009 resulted in infection of pregnant mares, none
of which transmitted infection to their foals based on
serial negative polymerase chain reaction (PCR)
results. Conversely, T. equi has been reported to be
responsible for 11% of all abortions in South Africa.60
Based on the variation in reported occurrence, it is
1337
likely that individual horse genetics or geographic iso-
late/strain differences could influence the prevalence of
placental transmission. Exposure to semen from an
infected stallion is not considered to be a means of
transmission, yet blood contamination during breeding
practices could present a transmission risk.61
In most cases, the horses become persistently
infected and become inapparent carriers. The inappar-
ent carrier state is life-long with T. equi and possibly
for B. caballi. A number of accounts indicate that
horses infected with B. caballi can undergo self-clear-
ance of the parasite without treatment.1,3,5 It is
assumed that persistent subclinical infection is due in
part to sequestration of the organism and immune
evasion strategies. Various theories for location of par-
asite sequestration in asymptomatic horses have been
reported, including capillaries, central nervous system
vasculature, and bone marrow.4,62,63 The mechanism(s)
of persistent infection remain unknown.
After transmission, depending on factors including
parasite dose and immunity, clinical signs develop
within 10–30 days for B. caballi and 12–19 days for
T. equi.3 The fatality rate of na€ıve horses in endemic
nations has been estimated at 5–10% dependent on
parasite, transmission dose, overall health of the horse,
and administration of treatment.4 Uniformly, infection
with T. equi typically results in more severe clinical
disease than B. caballi.4 Disease signs and severity can
vary significantly from 1 region to another.7,31
Clinical Disease
Clinical disease can manifest in different forms. With
acute T. equi infection, clinical signs are usually related
to marked hemolysis and resulting anemia. Although
B. caballi-infected horses do become anemic as well,
the rare cases of acute death from B. caballi have
occurred reportedly as a result of multiple organ
dysfunction related to systemic formation of micro-
1338 Wise et al
thrombi and development of disseminated intravascu-
lar coagulation.56 In these cases, the exact clinical signs
vary depending on the organ system affected.
Horses with acute infection initially develop nonspe-
cific signs such as high fevers, sometimes in excess of
104°F, lethargy, anorexia, weight loss, and peripheral
edema.4 Petechiations caused by thrombocytopenia are
often observed on mucous membranes, including the
nictitating membrane.3 Signs of hemolytic anemia fol-
low and include icteric or pale mucous membranes,
tachycardia, tachypnea, weakness, and pigmenturia
(because of either hemoglobinuria or bilirubinuria).53,64
Some horses show signs of gastrointestinal complica-
tions including colic or impactions followed by diar-
rhea. Other less common clinical presentations include
secondary development of pneumonia, pulmonary
edema, cardiac arrhythmias, catarrhal enteritis, lamini-
tis, and central nervous system disease characterized
by ataxia, myalgia, and seizures.32,39,64,65 Temporary
or permanent infertility has been reported in stal-
lions.56 Acute renal failure occurs as a result of hemo-
globin-induced pigment nephropathy and systemic
responses to severe inflammation (hypotension) can
worsen the kidney disease.3 Severe infections can also
culminate in liver failure or disseminated intravascular
coagulation.56
Fulminant, abrupt onset of signs of disease, termed
peracute disease, has been documented. Collapse and
sudden death from overwhelming T. equi can occur and
introduction of na€ıve horses into an endemic region can
lead to rapid onset of severe disease. In the 1930s, relo-
cation of a group of na€ıve horses into an endemic area
of southern France resulted in a 69% fatality rate.26 A
report from Jordan documented 5 presumed inapparent
carriers undergoing strenuous exercise that immediately
after completion of the exercise, developed profound
weakness with two dying suddenly.66 Recrudescence of
marked T. equi parasitemia was assumed. Neonatal
foals infected in utero with T. equi can present with
acute, severe signs.57,58,67–71 These foals can exhibit clin-
ical signs at birth or can become ill at 2–3 days of age.
Clinical signs are often nonspecific, such as weakness
and decreased suckling, but progress to resemble those
of an infected adult, including icterus, fever, and anemia
(with or without petechiations and hemoglobuinuria).
Cases of B. caballi fetal and neonatal infection have
been reported but are rare.70,72
Chronic T. equi or B. caballi infection can result
only in nonspecific signs, including lethargy, partial
anorexia, weight loss, and poor performance. Mild
anemia might be present and the spleen might be
enlarged upon rectal palpation. It has been suggested
that splenic enlargement is caused by the increased rate
of extravascular hemolysis that occurs within the
spleen in less severely affected horses.3,5,52
Importantly, horses infected with either T. equi or
B. caballi in both endemic and non-endemic regions
are most commonly inapparent carriers with no appre-
ciable signs of disease. Pregnancy in carrier mares can
result in abortion or neonatal infection.15,32 Because
inapparent carriers can serve as reservoirs for trans-
mission via ticks, placentally or iatrogenically, these
horses represent the largest challenge to non-endemic
nations attempting to prevent apparently healthy carri-
ers from crossing their borders.20,73
An appropriate list of differential diagnoses should
be determined based on whether the horse resides in or
has visited an endemic region. In general, acute onset
of the aforementioned clinical signs could also be
caused by equine infectious anemia virus, African
horse sickness virus, equine viral arteritis virus, equine
ehrlichiosis, purpura hemorrhagica, immune-mediated
hemolytic anemia, and red maple leaf toxicity.8,74
Results of laboratory analyses may aid in diagno-
sis. Most horses regardless of clinical syndrome exhi-
bit some degree of anemia characterized by decreased
packed cell volume, hemoglobin, and erythrocyte
count. Although acutely infected horses can have
profound anemia with packed cell volumes (PCV) as
low as 10%, the PCV rarely falls below 20%.52,54,64
Red blood cell indices, MCV, MCH, and MCHC are
variable.53 Thrombocytopenia is commonly identi-
fied.3,52,54,55,64 One report noted a decrease in platelet
count in 39% of T. equi infections, 80% of B. caballi
infections, and 100% of dual infection.54,64,75 Clotting
times can be prolonged or normal. The leukogram
can vary depending on infection stage and severity.76
Fibrinogen concentration can be elevated and albu-
min concentration can vary depending on hydration
status, chronicity of the disease, and associated con-
ditions that result in protein loss.55 Hyperbilirubin-
emia is often observed and the liver enzyme
activities, ALP, AST, and GGT can be elevated.64
These elevations are attributed to reduced blood flow
to the liver, which can in severe cases result in centri-
lobular necrosis. Hypophosphatemia and hypoferremia
are common, attributed to altered erythrocytic metabo-
lism.77 Infected erythrocytes can be identified in sternal
bone marrow aspirates of asymptomatic horses, but util-
ity of this test as a diagnostic tool is limited.62
Gross and histopathologic findings at necropsy vary
depending on the severity of disease and associated com-
plications. Gross examination might demonstrate evi-
dence of anemia as well as varying degrees of icterus,
edema, and splenomegaly. Other findings can include
pulmonary edema and congestion, cardiac hemor-
rhages, hydropericardium, hydrothorax, hepatomegaly,
ascites, enlarged discolored kidneys, and lymphadenop-
athy.3 Histopathologic findings can include centrilobu-
lar necrosis of the liver, renal tubular necrosis with
hemoglobin casts, and microthrombi within the liver
and lungs.32 Pulmonary tissue examination can demon-
strate congestion, edema, and hemosiderin-laden mac-
rophages within the pulmonary alveolar walls.
Parasites can be observed within red blood cells within
blood vessels and within macrophages in the lymph
nodes.3,39,78
The apparent global variation in clinical disease
might be caused by a variety of factors contributing to
emergence of infection and disease. Infection with
T. equi in South Africa often results in severe disease,
requiring treatment.31,32 In contrast, in the outbreak
 Equine Piroplasmosis
identified in the United States, in which 475 horses
were affected, only 1 horse was reported to exhibit
mild clinical signs.7 It is difficult to compare these
instances because the differences between these T. equi
strains are unknown, but it at minimum provides a
single comparison between non-endemic and endemic
countries.
Immunity
The response of the equine immune system to infec-
tion with T. equi or B. caballi is not completely
defined, but is undoubtedly complex and multifaceted.
It is well accepted that infection with either parasite
results in carrier status, which confers protection
against disease. There is no documented cross-protec-
tion between T. equi and B. caballi, as horses can be
infected with both parasites simultaneously.4
The spleen plays a necessary role in control of most
hemoprotozoan parasites. A horse with a spleen is typ-
ically able to overcome acute T. equi-induced disease,
whereas splenectomized horses invariably succumb to
disease with parasitemias that can reach 80%.51,53,79
Inapparent carriers of T. equi will also develop termi-
nal disease upon splenectomy.80 Splenectomized horses
inoculated experimentally with B. caballi may or may
not develop obvious clinical disease, but death caused
by infection has been documented.81 This discrepancy
may be attributable to differences in parasite strain,
infective dose, the overall health of the horse, or a
combination of factors.
Although essential in other hemoprotozoan infec-
tions like Babesia bovis, the function of cell-mediated
immunity in piroplasmosis has yet to be fully deter-
mined.82,83 Production of nitric oxide by macrophages
might be an essential effector mechanism of immune
control against experimental B. caballi infection.84
Importantly, innate immune responses and the pres-
ence of a spleen are not sufficient to control T. equi
infection, because spleen-intact foals with severe com-
bined immunodeficiency (SCID) are unable to control
T. equi parasitemia85 (Fig 3). Although innate immu-
nity is unaffected, SCID foals lack functional T and B
lymphocytes and are incapable of mounting antigen-
specific antibody and cellular immune responses.85–91
Inoculation of these foals with T. equi resulted in ful-
minant, severe infection within 7 days, and subsequent
death. Terminal parasitemias ranged between 29 and
41% and PCV decreased by 50% of the normal value.
Thus, adaptive immune responses are required to
control T. equi parasitemia, but are not required for
lysis of erythrocytes.
Antibody responses correlate with control of parasit-
emia.92 T. equi-infected horses produce antibodies
against immunodominant merozoite proteins termed
equi merozoite antigens (EMAs), which are surface
expressed on merozoites.85 The exact role of this anti-
body response in immunity and persistence remains
unclear. The nomenclature of equine immunoglobulin
G has recently been adjusted to accommodate the 7
unique IgG heavy chain genes discovered in the equine
1339
Fig 3. Electron micrograph of a splenic macrophage from a
severe combined immunodeficiency foal showing phagocytized
Theileria equi-infected erythrocytes. Arrow denotes the organism
inside an erythrocyte and the asterisk illustrates the nucleus of
the splenic macrophage. Image courtesy of Lowell Kappmeyer.
genome.93 In the acute stages of T. equi infection, high
levels of IgGa (now IgG1) and IgGb (now IgG4&7)
correlate with control, whereas IgG(T) levels (now pre-
dominately IgG5 and to a lesser extent IgG3) increase
after resolution of parasitemia during the chronic
phase of infection.92 Antibodies are first detected
within 7–11 days after natural infection and peak at
30–45 days. All examined subclasses remain detectable
into the chronic/inapparent stage of disease.
The correlates of adaptive immune responses to
T. equi infection are currently unknown. Donkeys vac-
cinated with T. equi immunogen were able to mount a
protective response characterized by high antimerozo-
ite antibody titers and merozoite-specific lymphocyte
proliferation.83 Recently, it was documented that
SCID foals that received repeated infusions of T. equi
hyperimmune plasma before inoculation with the para-
site were able to delay the time to peak parasitemia.94
All foals developed disease, but partial protection was
associated predominantly with transfused IgG3-specific
antimerozoite antibodies. Overall, additional research
is needed to define the protective roles of antibody and
cellular adaptive immune responses against T. equi.
Even less is known about protective immune
responses against B. caballi infection. Infected horses
produce antibodies to rhoptry associated protein-1
(RAP-1), which is utilized on serologic detection of
infection. This conserved apical merozoite protein
remains partially uncharacterized in B. caballi, but in
B. bovis plays a pivotal role in induction of humoral
immunity.95
In most endemic areas, foals that ingest colostrum
from a carrier mare are protected from infection and
clinical disease for the first 1–5 months and can be
protected up to 9 months of age.3,56 As maternal anti-
bodies decline, the foal becomes susceptible and most
1340
young horses in endemic nations are infected by age 2.
It has also been suggested that foals can be born as
healthy inapparent carriers of T. equi, which would
also infer some level of protection.56
Rarely, inapparent T. equi carriers can exhibit
relapses of clinical disease associated with stress, stren-
uous exercise, immunosuppression, and steroid
administration.66,96,97 Experimental treatment with
beclamethasone at a dose of 0.1 mg/kg once daily for
5 days before and 5 days after inoculation with T. equi
resulted in a 50% increase in parasitemia as compared
with controls.96 These relapses have not been reported
for B. caballi.
Diagnosis
Various diagnostic modalities can be used alone or
in combination to diagnose infection. During manage-
ment of an outbreak within a non-endemic nation,
involvement of the state and national regulatory agen-
cies is required and often, multiple diagnostic methods
will be utilized in an effort to obtain the most accurate
information. Only a few laboratories in the world are
authorized to perform certain tests, so proper handling
of samples is crucial.
Light microscopy can be used to identify the organ-
isms within the erythrocytes. A thin blood smear,
stained with Giemsa, Wright’s, or Diff-Quik®, may
reveal organisms during the acute stage of infection.
The smears must be thoroughly examined since even
during severe infection, the percent parasitemia
remains so low that false-negative results are not
uncommon.5,98 The piroplasms of T. equi and B. caballi
can be easily distinguished from one another. Within the
erythrocyte, B. caballi typically appears as 2 large pyri-
form (pear-shaped) merozoites that measure approxi-
mately 2–5 lm in length (Fig 4). During clinical infection
with B. caballi, the percentage of erythrocytes parasitized
is typically less than 1% and may be less than 0.1%.
T. equi merozoites occur within erythrocytes as poly-
morphic, small piroplasms occasionally in a distinct
Maltese cross-formation (Fig 5). The T. equi merozo-
Fig 4. Equine erythrocyte containing Babesia caballi merozoites.
Diff-Quik®, 9100 oil magnification. Image courtesy of Peter
Awinda.
Wise et al
ites are smaller and typically measure 2–3 lm in
length.3 The percent of infected erythrocytes during
clinical disease caused by T. equi is usually between 1
and 5%, but in severe cases can exceed 20%.56 In
cases of chronic or inapparent infection, parasite num-
bers remain too low for reliable detection on blood
smear.99
Several serologic tests were developed to increase
diagnostic sensitivity, especially in those carrier horses
exhibiting no clinical signs. These tests include the
CFT, indirect immunofluorescence assay, western blot,
and competitive enzyme-linked immunosorbent assay.
The CFT relies on activation of complement upon
specific interaction of antibody and antigen.99 A posi-
tive result is defined as a positive reaction at a dilution
of 1 : 5. Infected horses seroconvert on CFT approxi-
mately 8–11 days after infection with titers beginning
to decline at 2–3 months.99 CFT is a very specific test,
yet lacks sensitivity, especially in chronic infection or
after treatment.27 Horses can transiently become nega-
tive 3–15 months after treatment for B. caballi and
24 months for T. equi.99,100 IgG(T), now classified as
IgG5 and to a lesser extent IgG3, remains elevated in
chronic T. equi infections. IgG(T) does not fix comple-
ment and recently, it was demonstrated that IgG3 fixes
complement, whereas IgG5 does not.93,101 Thus, it is
not surprising that the CFT lacks sensitivity for diag-
nosis of chronic or inapparent T. equi infection. Cross-
reactivity between antibodies against T. equi and
B. caballi when using the CFT has been reported.99,102
Regardless of the fact that the CFT was previously the
official regulatory test for establishing piroplasmosis
status before travel to a non-endemic country, the
CFT is not considered the diagnostic test of choice for
chronic infection.98
Indirect immunofluorescent antibody tests (IFAT)
demonstrates high specificity, but lacks sensitivity. It
is, however, considered more sensitive than the CFT.99
In this test, fluorescently labeled antibodies react with
antigen bound to a glass slide. A sample is considered
positive if strong fluorescence is noted at a dilution of
1 : 80 or higher. Experimental intravenous infection
Fig 5. Equine erythrocyte containing Theileria equi merozoites
in characteristic Maltese cross-formation. Diff-Quik®, 9100 oil
magnification.
 Equine Piroplasmosis
with T. equi or B. caballi resulted in positive IFAT
results at days 3–20 post infection.100 Titers were more
consistently detected and remained elevated longer
with the IFAT versus the CFT.103 Typically, 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.98
Until recently, Western blot (or immunoblot) has
been utilized primarily in a research setting for diagno-
sis of T. equi and B. caballi. The National Veterinary
Services Laboratory in Ames, Iowa, is now offering an
immunoblot as an adjunct diagnostic tool for detection
of B. caballi infection.104 Research is under way to val-
idate these tests for use in routine diagnosis.
Since 2004, the competitive inhibition enzyme-linked
immunosorbent assay (cELISA) has been one of the
regulatory tests prescribed by the OIE for international
horse transport.98 The test is considered to be the most
sensitive means of detection of chronic T. equi infec-
tion.27 The cELISA for T. equi utilizes recombinant
EMA-1 and specific monoclonal antibodies.105 EMA-1
is an immunodominant, highly conserved surface anti-
gen specific to T. equi. Horses infected with T. equi are
detectable with the cELISA as early as 21 days after
experimental infection and approximately 5 weeks
after tick transmission.105 The EMA-1 cELISA is vali-
dated for use against multiple different strains of
T. equi found around the world.27 Generation of a
recombinant form of this epitope and associated
monoclonal antibodies allowed standardization of this
test and markedly increased sensitivity as compared
with the other serologic tests.27,106,107
A recombinant form of RAP-1 was also developed
for the B. caballi cELISA.108 This test, upon compari-
son with CFT using 300 equine serum samples from
around the world, was able to diagnose infection in
25% more cases than with the CFT. However, the
currently available RAP-1 cELISA relies on the recog-
nition of epitopes that are not conserved across all
B. caballi strains. Because of sequence heterogeneity
between the recombinant RAP-1 used in the test and
South African isolates of B. caballi, the test is unable
to detect infected horses in South Africa.109 Although
similar differences exist between the recombinant
EMA-1 used in the T. equi cELISA and South African
T. equi isolates, the cELISA continues to detect
infected horses in South Africa.110 Both cELISAs are
marketed by VMRD (Pullman, WA) and are not
available to general practitioners. Lastly, a field vali-
dated ELISA using whole T. equi merozoite antigen
has been described as an easy, economical, and reliable
test.111
Recently, the previously reported data on the speci-
ficity and sensitivity of the CFT and cELISA were
statistically analyzed.25,106,108,112 Overall, the sensitivity
of the CFT to detect T. equi is 47% and the cELISA
is 96%. The specificities of the 2 tests are 94% and
95%, respectively. For testing of B. caballi, the sensi-
tivity of the CFT is 88% and the cELISA is 91%. The
specificities of the 2 tests for B. caballi are 98% and
70%, respectively.
1341
Polymerase chain reaction tests for the presence of
the organism of interest by amplifying and detecting
specific fractions of the DNA. This test is exquisitely
sensitive and thus far has only been utilized in research
settings for the detection of T. equi. Three variations
of PCR include real-time PCR, nested PCR, and
nested PCR with hybridization.113–117 Comparison of
these tests and the results from these tests is difficult,
given that no standardization exists between laborato-
ries. Nested PCR for T. equi using the EMA-1 gene
sequence has been shown to detect a positive result
equivalent to a percent parasitemia of 0.000006%.115
One report also indicated that nested PCR detects
3.69 more infections that microscopy and 2.29 more
than traditional PCR.30 The validity of nested PCR
routinely in the United States has been questioned as a
diagnostic tool for horses in South Africa.110,118 Upon
examination of the genetic composition of EMA-1, it
was recognized that the strains from around the world
were not 100% homologous. These discoveries further
emphasize the issues involved in standardization of
PCR as a diagnostic test. Thus far, the use of nested
PCR for detection of B. caballi DNA in chronically
infected horses has proven unreliable. As it is currently
performed, it is unlikely that nested PCR will ever be
standardized and commercially marketed for detection
of T. equi or B. caballi infection.119
With increasing use of PCR in laboratory settings,
the validity of a positive cELISA result has come into
question. Despite treatment and apparent clearance of
T. equi, as demonstrated by negative PCR and trans-
mission studies, cELISA results remain positive, some-
times for up to 24 months after clearance.7,120 The
transmission risk that these PCR-negative, seropositive
horses pose must be determined.
Treatment
In endemic regions, treatment of piroplasmosis is
used only as a means of decreasing clinical signs and
reducing fatalities. Clearance of the organism serves
no purpose in these countries as life-long immunity
(premunity) is assumed to be conferred with chronic,
inapparent infection. In non-endemic regions attempt-
ing to remain free of piroplasmosis, treatment of
infected horses with the intent of clearance (chemoster-
ilization) is desired. T. equi infections are more typi-
cally difficult to treat than B. caballi infections.
Numerous drugs have been reported to have variable
efficacy in inhibiting T. equi and B. caballi both in cell
culture and in vivo, making the literature difficult to
interpret.117,121–128 Historically, it was reported that
B. caballi infection was self-limiting with clearance
noted after several years, yet this is not always the
case.63 Chemotherapeutic clearance of T. equi in the
horse has been previously reported, yet the research
was conducted before the development of tests with
increased sensitivity for parasite persistence.122,123
Until recently, it was widely accepted that chemosteril-
ization of a T. equi-infected horse was unachievable.
Data collected during the outbreak in Texas indicate
1342
that T. equi can be eliminated from an infected horse
with appropriate dosing of imidocarb diproprionate.7
For alleviation of clinical signs, several drugs have
been used with success, yet imidocarb, in its diproprio-
nate salt form (ID), is considered to be the most effective.
The alternate form of this drug, composed of dihydro-
chloride salt, will cause more severe muscle damage at
the site of injection.122,129 ID, a carbanilide derivative, is
typically administered to horses intramuscularly.
Although its mechanism of action remains unclear, pro-
posed mechanisms include inhibition of inositol entry
into infected erythrocytes or alteration in metabolism of
polyamines.130,131 After intramuscular (IM) injection,
ID is rapidly eliminated from the plasma, yet remains
sequestered in certain tissues of the body.132 Reported
dosages for alleviation of clinical signs vary, yet most
sources indicate that 2.2–4.4 mg/kg given IM once is
effective. If necessary, lower dosages can be repeated
at 24–72 hour intervals for 2–3 treatments. In non-
endemic nations where chemotherapeutic clearance of
the organism is desired, animals infected with B. cabal-
li can be cleared with a dose of 4.4 mg/kg IM every
72 hours for 4 treatments.117 For clearance of T. equi,
data from both naturally and experimentally infected
horses indicate that the same dose is effective.7,120 Of
the 25 naturally infected treated horses, one remained
positive for T. equi after the initial treatment and had
to undergo a 2nd treatment regimen to obtain chemo-
sterilization.7 In the study using ID to attempt clear-
ance in experimentally T. equi-infected horses, 1 horse
remained resistant to parasite elimination.120 Cur-
rently, clearance is determined by negative PCR results
and inability to transmit the parasite to a na€ıve sple-
nectomized horse via blood transfusion, but studies
are under way to more clearly define parasite elimina-
tion.7 In the United States, if a horse is diagnosed with
T. equi, the owner and veterinarian can enroll the
horse in the USDA controlled treatment program as
to ensure appropriate quarantine, treatment, and sub-
sequent release of cleared horses.120
ID has anticholinesterase activity, so reactions to the
drug often present as sweating, signs of agitation, colic,
and diarrhea.129,133 Typically, these signs are transient
and rarely life-threatening. Effects can be prevented with
an intravenous dose of glycopyrolate at 0.0025 mg/kg
once or reversed with a single intravenous dose of atro-
pine at 0.2 mg/kg. Both of these anticholinergic drugs
cause adverse effects as well. Administration of the
anticholinergic n-butylscopolaminea can lessen clinical
signs without addition of adverse effects.7 As ID
undergoes hepatic and renal clearance, periportal hepa-
tic necrosis and renal tubular necrosis can occur with
toxicity.129,133 Horses undergoing treatment with this
drug should be monitored carefully for development of
complications. Transient azotemia, elevations in uri-
nary GGT/creatinine ratios, or both as well as tran-
sient elevations in liver enzyme activities (AST, ALT,
ALP, and SDH) can be observed during treatment,
but typically resolve with discontinuation of the
drug. Donkeys and mules are exquisitely sensitive
to ID, therefore its use in these species is not
Wise et al
recommended.122 Information regarding treatment of
T. equi-infected neonatal foals with ID is limited with
only one case report.71 When a nursing mare is given a
single dose of at 2.4 mg/kg IM, ID is detectable in the
milk 2 hours after administration.132 It remains
unclear if this could lead to toxicity in the suckling
foal. One report of administration of ID to pregnant
mares followed by induced abortion resulted in circu-
lating levels of ID in each fetus comparable to the
dam’s serum concentrations.60
Diminazene aceturate and diaminazene diaceturate
have been used with success against T. equi and
B. caballi at a dose of 3.5 mg/kg IM every 48 hours
for 2 treatments.134 Diminazene aceturate is more
effective than diaminazene diaceturate, but both drugs
have been reported to cause significant injection site
muscle damage. Efficacy of both drugs increases with
the 2nd dosage, yet no chemosterilization has been
reported. Signs of toxicity include respiratory distress
and lethargy.
The antibiotic oxytetracycline when administered IV
at a dose of 5–6 mg/kg once daily for 7 days is effective
against T. equi, but not against B. caballi.64 Other drugs
reported to have efficacy in treatment of babesiosis
include amicarbilade isothionate, euflavine, artesunate
and arteether (arteminsin derivatives), buparvaquone,
and atovaquone, yet these drugs are no longer com-
monly utilized in practice.56,124,125 Ponazuril inhibits
T. equi organisms in vitro.135 No in vivo research has
been conducted to date, yet it is possible that this class
of drugs could offer additional treatment options in
the horse.
Aside from antiprotozoal drugs, acutely infected
horses often require supportive care including but not
limited to intravenous fluids, nonsteroidal anti-inflam-
matory drugs, pain management, and blood transfu-
sions. Adequate hydration is essential upon initiation
of and during treatment with imidocarb.
Prevention/Vaccination
Prevention of infection in endemic nations is virtu-
ally impossible, and it is assumed that the premunity
conferred with initial infection acts to protect the horse
from recurrent disease upon subsequent exposures. In
non-endemic nations, the cornerstone of protection is
regulation of equine movement between endemic
nations. Depending on the non-endemic country in
question, horses must test negative for T. equi and
B. caballi on the serologic test designated specifically
by the country of import, typically the cELISA or the
IFAT. If positive, horses are generally denied entrance
unless for tightly regulated athletic events. All
imported horses from endemic nations undergo strict
quarantine and are examined thoroughly for ticks.
Application of acaricides before removal from the
endemic nation is used to ensure that ticks are not
introduced with the horses. The regulatory system put
in place by the OIE has been successful, yet isolated
cases continue to occur in non-endemic nations. These
isolated cases are rarely caused by tick transmission
 Equine Piroplasmosis
and are most often linked to the use of blood contami-
nated equipment and practices involving needle sharing
or blood transfusions from untested donors.37,39,78 In
the United States, a horse that is identified as positive
on cELISA must be immediately quarantined and the
state and federal authorities must be notified. Until a
reliable means of sterilization is identified, these horses
must remain quarantined, be exported, donated to
research facilities, or euthanized. The most appropriate
action is determined by the state and federal regula-
tory officials on a case-by-case basis.25,120
Non-endemic nations that border endemic nations
cannot completely prevent introduction of ticks, so dil-
igent measures must be taken to reduce horses’ contact
with ticks. This includes routine application of acari-
cides, surveillance for the presence of ticks, and
reduction in vegetation.73 A variety of chemical are
available to reduce tick exposure, but not all effective
acaricides conform to EPA regulations, so careful
selection and use of these products is necessary.25
Although no direct scientific evidence exists in horses,
systemic administration of the dewormer ivermectin
likely aids in control of blood-feeding ticks. Being
aware of the habitat and the seasonality of ticks in
your area is important. Guidelines for examining
horses for ticks and appropriate removal of ticks have
been published by the USDA.73
Several studies have assessed the potential use of
vaccination to induce immunity to B. caballi and
T. equi infection, yet no vaccine is commercially
available. Immunization with whole merozoite induced
protection in 4 donkeys.83 The correlates of protective
immunity in horses are unknown, and until these are
elucidated, developing an effective vaccine will be
difficult.
Vaccination and treatment strategies are dependent
on the infection status (endemic versus non-endemic) of
the region. A transmission blocking vaccine in the
absence of full compliance in at-risk horses would not
be appropriate in an endemic region. A vaccine prevent-
ing transmission or clinical disease and death is needed
for na€ıve horses moved into endemic regions. Chemo-
therapeutics, which aid in control of acute parasitemia
and associated clinical signs, but do not eliminate
infection, are important in endemic regions. In non-
endemic regions, the goal is remaining infection free;
therefore, when infected horses are detected, a safe
chemotherapeutic with efficacy for eliminating persis-
tent infection is needed. To remain infection free, nec-
essary tools include accurate screening diagnostic tests
and thorough knowledge of tick populations and their
ability to transmit B. caballi, T. equi, both.
Increasing globalization of the equine industry cou-
pled with constantly changing climates provides major
challenges for controlling persistent infections as those
caused by T. equi and B. caballi. As with all vector-
borne infections, measures must be taken to enhance
control through surveillance of equine populations and
detailed knowledge of vector competence and habitat.
Detailed information regarding life cycles, transmis-
sion, immune responses, accurate diagnostics, and
1343
treatment of these parasites is paramount to control
these insidious diseases, while promoting growth in the
movement of horses internationally.
Footnote
a
Buscopan; Boehringer Ingelheim, Rheinland-Pfalz, Germany
Acknowledgments
Conflict of Interest Declaration: The authors disclose
no conflict of interest.
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