pathogens Review Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny Sharon Tirosh-Levy 1,* , Yuval Gottlieb 1, Lindsay M. Fry 2,3, Donald P. Knowles 2 and Amir Steinman 1 1 Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot 7610001, Israel; [email protected] (Y.G.); [email protected] (A.S.) 2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA; [email protected] (L.M.F.); [email protected] (D.P.K.) 3 Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, WA 99164, USA * Correspondence: [email protected] Received: 7 September 2020; Accepted: 4 November 2020; Published: 8 November 2020 Abstract: Equine piroplasmosis (EP), caused by the hemoparasites Theileria equi, Theileria haneyi, and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. Infection may affect animal welfare and has economic impacts related to limitations in horse transport between endemic and non-endemic regions, reduced performance of sport horses and treatment costs. Here, we analyzed the epidemiological, serological, and molecular diagnostic data published in the last 20 years, and all DNA sequences submitted to GenBank database, to describe the current global prevalence of these parasites. We demonstrate that EP is endemic in most parts of the world, and that it is spreading into more temperate climates. We emphasize the importance of using DNA sequencing and genotyping to monitor the spread of parasites, and point to the necessity of further studies to improve genotypic characterization of newly recognized parasite species and strains, and their linkage to virulence. Keywords: equine piroplasmosis; Theileria equi; Babesia caballi; equine; genotyping 1. Current Knowledge of Equine Piroplasmosis Equine piroplasmosis (EP) is a tick-borne disease of equines caused by the eukaryotic hemoparasites Theileria equi, Theileria haneyi, and Babesia caballi that has a considerable veterinary and economic impacts on the horse industry worldwide [1–5]. The parasites belong to the phylum Apicomplexa and to the order Piroplasmida [6]. EP is considered a reportable disease by the World Organization for Animal Health (OIE) (https://www.oie.int/animal-health-in-the-world/oie-listed-diseases-2020/, 15 April 2020). It is estimated that 90% of the global horse population resides in EP-endemic areas, and therefore many studies have investigated the occurrence, prevalence, risk factors, and characteristics of these parasites in different parts of the world. 1.1. Life Cycle, Vectors, and Transmission The Theileria and Babesia genera belong to the families Theileriidae and Babesiidae within the phylum Apicomplexa. The life cycles of both parasites include sexual (gamogony) and asexual (sporogony) replicative stages within the tick vector and asexual replicative stages within the equine host [2,3]. Asexual replication (merogony) in equine erythrocytes is common to both parasites, Pathogens 2020, 9, 926; doi:10.3390/pathogens9110926 www.mdpi.com/journal/pathogens Pathogens 2020, 9, 926 2 of 32 Pathogens 2020, 9, x 2 of 35 and T. equi (and likely, T. haneyi) also undergoes asexual schizogony within equine lymphocytes and haneyimonocytes) also undergoes prior to invasionasexual schizogony to erythrocytes within [7 ]equine (Figure lymphocytes1). The term and piroplasmosis monocytes prior derives to invasion from the to erythrocytespear-shaped [7] appearance(Figure 1). The of the term intra-erythrocytic piroplasmosis de stagesrives from of these the pear-shaped parasites (merozoites). appearance Replicationof the intra- erythrocyticin erythrocytes stages ultimately of these parasites leads to cell(merozoites). rupture and Replication the release in of erythrocytes merozoites thatultimately invade leads additional to cell rupturecells [ 2and,3,8 ].the release of merozoites that invade additional cells [2,3,8]. FigureFigure 1. The 1. The life lifecycle cycle of Theileria of Theileria equi equi (TE)(TE) and and BabesiaBabesia caballi caballi (BC)(BC) in the in tick the tickvector vector and andin the in equine the equine host. RBC—equinehost. RBC—equine red blood red cells, blood WBC—equine cells, WBC—equine while blood while cells, blood SG—tick cells, salivary SG—tick glands. salivary glands. TheThe main main route route of of transmission transmission to to equids equids is is by by tick tick feeding. Over Over 30 species ofof ticksticks havehave beenbeen describeddescribed as asvectors vectors of of one one or or both both T.T. equi equi and B. caballicaballi,, includingincluding thethe generageneraHyalomma Hyalomma,,Rhipicephalus Rhipicephalus DermacentorDermacentor, Amblyomma, Amblyomma, and, andHaemaphysalisHaemaphysalis [8]. Transstadial[8]. Transstadial transmission transmission was recorded was recorded for both forparasites both in parasitesseveral tick in severalspecies; tick however, species; transova however,rian transovarian transmission transmission was only recorded was only for recorded B. caballi for [8].B. Therefore, caballi [8]. theTherefore, main reservoir the main for reservoirT. equi is in for theT. equiequineis in host, the whilst equine for host, B. caballi whilst it for is B.the caballi vectorit ticks is the [8]. vector ticks [8]. TransplacentalTransplacental transmission transmission in the in equine the equine host has host been has reported been reported for T. equi for andT. equi mayand lead may to abortion, lead to theabortion, birth of thea sick birth foal of with a sick peracute foal with neonatal peracute EP, neonatal or the EP,birth or theof unapparent birth of unapparent carrier foal carrier [9–19]. foal In [9 some–19]. endemicIn some areas, endemic T. equi areas, is consideredT. equi is considered to be a major to cause be a major of abortion cause of[20,21]; abortion however, [20,21 ];the however, role of this the parasite role of as thisa cause parasite of abortion as a cause is not of well abortion established is not well [22]. established Iatrogenic transmission [22]. Iatrogenic is also transmission possible; there is also are possible; several reportsthere of are infections several reports resulting of infectionsfrom blood resulting transfusions, from and blood from transfusions, sharing of surgical and from equipment sharing of or surgical needles [2,3,5,20].equipment However, or needles these [ 2types,3,5,20 of]. transmission However, these probab typesly do of transmissionnot have a major probably role in do the not epidemiology have a major of EP.role in the epidemiology of EP. 1.2.1.2. Clinical Clinical Disease Disease ClinicalClinical disease disease in inEP EP is ismainly mainly attributed attributed to to intravascular intravascular hemolytic anemia causedcaused byby parasiteparasite replicationreplication and and damage damage to toerythrocytes erythrocytes [2,3,20,23]. [2,3,20,23 The]. Theclinical clinical signs signs are similar are similar following following infection infection with bothwith parasite both parasite species; species;however, however, clinical presen clinicaltation presentation tends to tends be more tobe severe more in severe cases inof casesT. equi of infectionT. equi [2,3,20].infection The [ 2incubation,3,20]. The period incubation ranges period between ranges 12 betweenand 19 days 12 and for 19T. daysequi and for T.between equi and 10 between and 30 days 10 and for B. 30caballi days [2]. for CommonB. caballi clinical[2]. Common signs are clinical non-specific signs are and non-specific derive from and the derive hemolytic from theanemia. hemolytic These anemia. include These include fever, inappetence, icterus, hemoglobinuria, pale mucus membranes (MM), tachycardia, and tachypnea. Thrombocytopenia has also been described. In severe cases, edema and hemorrhage might develop and may eventually lead to organ failure. Gross pathologic findings may include hepatomegaly, Pathogens 2020, 9, 926 3 of 32 splenomegaly, enlarged kidneys, multifocal edema, and hemorrhages [2,3,20,23]. Anecdotal cases of EP-associated hyphema [24], cardiac arrythmias [25], and inflammatory myopathy [26] have also been reported. Clinical manifestations following infection with either parasite species range from unapparent infection to life threatening disease. Most infected horses remain asymptomatic [2,3,20], while clinically infected horses may develop peracute, acute, subacute, or chronic disease presentations. Peracute disease is life-threatening and has been mostly described in cases of neonatal EP [2,3,20]. Acute disease is characterized by overt presentation of characteristic EP clinical signs, subacute disease manifests milder clinical signs, and chronic disease presents with non-specific signs and mild clinical pathology abnormalities [2,3,20,23,27]. The factors associated with the severity of clinical disease are unknown. Acute disease is more often observed in infections of naïve adult horses, and is less common in equine populations in endemic areas [2,3,20,23]. Stress has been suggested to induce more severe clinical signs, although the evidence to support this assumption is limited [28]. In contrast to T. equi, the newly identified T. haneyi rarely causes clinical signs, even in splenectomized horses [29]. Regardless of the initial clinical presentation, without treatment, horses infected with EP usually remain persistent subclinical (unapparent) carriers for prolonged periods of