Equine Piroplasmosis

Equine piroplasmosis (EP) is a tick-borne protozoal disease of horses, mules, donkeys, and zebras that is characterized by acute hemolytic anemia. The etiologic agents are two hemoprotozoan parasites, Theileria equi (Laveran, 1901) and Babesia caballi (Nutall and Strickland, 1910) that are transmitted primarily by ixodid ticks. Equine piroplasmosis is found globally where tick vectors are present and is endemic in tropical, subtropical, and some temperate regions. Horses infected with B. equi remain seropositive for life; horses infected with B. caballi are seropositive for several years to life. Economic losses associated with EP are significant and include the cost of treatment, especially in acutely infected horses; abortions; loss of performance; death; and restrictions in meeting international requirements related to exportation or participation in equestrian sporting events. Equine babesiosis–free countries limit the entrance of Babesia-seropositive horses into their countries. In the United States a few sporadic outbreaks have occurred in recent years but have been limited due to implementation of stringent control methods. The cELISA for both T. equi and B. caballi is currently the recommended test for international horse transport. Different therapies for control and sterilization of the parasites are discussed.     

Equine piroplasmosis: An updated review

Equine piroplasmosis (EP) is a tick‐borne protozoal disease. The causative agents are Babesia caballi and Theileria equi. Horses infected with T. equi remain carriers for life. Iatrogenic means can also be factors for transmission. Typical clinical signs of acute EP can include fever, anorexia, anaemia, icterus, congested mucous membranes, tachypnoea and tachycardia, sweating, and limb and supraorbital oedema. In severe cases, haemoglobinuria and bilirubinuria are present as well as a variety of atypical presentations due to organ damage and dysfunction. Because clinical pathology is not specific of EP, accurate diagnosis requires specific diagnostic tests. The value and the pertinence of blood smears, polymerase chain reaction and serological tests are presented. Imidocarb propionate is considered as the drug of choice against EP. However, treatment strategies differ greatly between endemic and nonendemic regions. In endemic regions the goal is to reduce clinical disease because premunition plays an important role in the protection of horses, while in nonendemic regions the goal of treatment is to eliminate the risk of transmission with sterilising treatment protocols. As there is no effective vaccine available to date, prevention relies mainly on drug therapy, restriction in the movement of infected horses, and control of tick vectors.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

Clinical and Laboratory Findings in Equine Piroplasmosis

The objective of this study was to evaluate equine piroplasmosis (EP) as a cause of morbidity in horses in Sardinia (Italy), describe the clinical signs and altered hematologic and biochemical parameters, and illustrate response to different treatments. Among 44 horses suspected of tick-borne disease, 38 were polymerase chain reaction (PCR) positive for Theileria equi (n = 27) or Babesia caballi (n = 6), whereas five were positive for both protozoans. Typical clinical features of piroplasmosis were seen in some of the horses, whereas others had nonspecific mild symptoms. Hematologic findings revealed involvement of the three blood cell lineages (anemia, leukopenia or leukocytosis, thrombocytopenia), and biochemical variations were related to increased bilirubin, alteration of serum phosphorus, and hypoalbuminemia. We suggest that the two protozoans are the most important causative agents of equine tick-borne disease in this geographic area, and we observe that different clinical features are associated with the disease; in addition to the typical aspects of piroplasmosis, characterized by fever, pale mucous membranes, and icterus, we can signal other nonspecific mild signs such as weight loss, weight loss associated with an insignificant leukopenia, or weight loss associated with depression, anorexia, and mild hyperbilirubin. The study is intended as a practical contribution for veterinary practitioners because it describes different clinical presentations and laboratory findings of EP, suggests diagnostic and therapeutic approaches to the disease, and shows diffusion of the disease in a Mediterranean region.     

The First Report of Serological Detection of Babesia caballi by cELISA in a Horse During Serological Survey of Piroplasmosis in Imported Horses at Shanghai Port,China

The aim of this study was to determine the seroprevalence of Babesia caballi and Theileria equi in horses imported into Shanghai port. Between 2018 and 2019, 344 horse sera samples were collected and tested for B. caballi and T. equi, using commercially available kits. Only one B. caballi seropositive sample was detected. To the best of our knowledge, this is the first report of a B. caballi seropositive in imported horses at Shanghai port, which reflects the importance of monitoring piroplasmosis seroprevalence in imported horses.     

Investigation of seroprevalence of <Emphasis Type="Italic">Theileria equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> in horses in Nigde province,Turkey

The prevalence of equine piroplasmosis caused by Theileria equi and Babesia caballi in Nigde, in central Anatolia, Turkey has remained unknown. Serum samples were obtained from a total of 125 horses and were tested for antibodies to T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). Twenty-three (18.4%) horses were seropositive for equine piroplasmosis. Anti-T. equi was observed in 16 horses (12.8%) while anti-B. caballi was detected in 12 horses (9.6%). In addition, 5 serum samples were positive for both parasites. The prevalence rates of antibodies to T. equi and B. caballi for female and male horses were statistically indifferent (p = 0.19 and 0.90). The difference between the seropositivity rates to T. equi among age groups was statistically insignificant (p = 0.44) while the difference to B. caballi among age groups is statistically significant (p = 0.01). Seropositivity rates ranged from 2.9% to 25.7% for T. equi and 2.9% to 14.3% for B. caballi from the selected districts in Nigde. A statistically significant difference on seropositivity rates for the study sites was observed for only T.equi (p = 0.03). This study indicates that T. equi is higher than B. caballi in Nigde. This study was supported by the Scientific Research Projects Unit of Nigde University (FEB 2007/08).     

Status of Lipid Peroxidation, Antioxidants, and Oxidation Products of Nitric Oxide in Equine Babesiosis: Status of Antioxidant and Oxidant in Equine Babesiosis

Equine babesiosis is a tick-borne protozoal disease of horses caused by Theileria equi and Babesia caballi. The disease is endemic in most tropical and subtropical areas. The aim of this paper is to assess the antioxidant status, lipid peroxidation, and oxidation products of nitric oxide (NO) in horses and mules naturally infected with T. equi and B. caballi. East and Southeast Anatolian horses and mules living in rural region of the Eastern border of Turkey were used as the material for this study. These animals are used as pack animal (3–7 years of age). Infected animals were in acute or subacute infection period. In the current study, malondialdehyde (MDA), oxidation products of NO (nitrate and nitrite), serum glutathione (GSH), vitamin E, and retinol levels were analyzed in 58 equids (horse and mule) infected with T. equi and B. caballi as well as in 44 healthy equids. Compared with controls, the level of MDA and nitrate increased significantly (P < .01, P < .05, respectively), whereas GSH concentration and levels of vitamin E decreased significantly (P < .01). There was no significant change in the level of nitrite and retinol between two groups. The results of the current study suggest that in equids infected with T. equi and B. caballi, this alteration in the lipid peroxidation, oxidants, and antioxidants may be related to the host's defenses against parasitic infection and may play a central role in the pathologic conditions associated with babesiosis.     

Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids

A cross-sectional study was carried out on equids (horses, mules and donkeys) in Andalusia, Southern Spain, to assess the level of exposure to equine piroplasmosis and to investigate risk factors associated with these infections. At least one animal seropositive for Theileria equi and/or Babesia caballi was detected in 222/380 (58.4%) herds sampled by competitive inhibition ELISAs. The seroprevalences for B. caballi and T. equi were 13.2% and 56.1%, respectively; there was serological evidence of co-circulation of both piroplasms in 10.8% of herds. Antibodies against equine piroplasms were detected in 286/537 (53.3%) animals; 61 (11.4%) were seropositive for B. caballi, 270 (50.3%) were seropositive for T. equi and 24 (8.4%) were seropositive for both T. equi and B. caballi.There was a significantly higher seroprevalence of B. caballi in mules (32.1%) compared with donkeys (17.0%) and horses (7.9%), and a significantly higher seroprevalence of T. equi in mules (66.1%) in comparison with horses (48.6%), but not donkeys (47.2%). There were significant differences in prevalence of both piroplasms among locations; the seroprevalence of B. caballi ranged from 0 to 22.5%, while the seropositivity to T. equi ranged from 26.7 to 63.3%. A multiple logistic regression model indicated that the risk factors associated with a higher T. equi seroprevalence were increased age, presence of ticks and vaccination against other diseases. Risk factors associated with a higher seroprevalence of B. caballi were species (mules compared to horses), entry of horses in the last 6 months, presence of ticks and presence of shelter. The findings indicate widespread exposure to equine piroplasmosis in Southern Spain.     

Review of Equine Piroplasmosis

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 parasites are able to persist indefinitely in their equid hosts, thus far, only B. caballi transmits across tick generations. Pathogenesis 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 factors leading to the re‐emergence of infection and disease of these globally important pathogens.     

Seroprevalence and risk factors associated with Equine piroplasmosis in North Egypt

Equine piroplasmosis (EP) is caused by Theileria equi and/or Babesia caballi and has economic importance particularly in equines reared in poor management systems. This study is based on cELISA test to study the seroprevalence of EP among 370 horses and 150 donkeys in four Governorates north Egypt. Additionally, its risk factors were studied for the first time. The seroprevalence rates 36.5 %, 20 %, and 5.6 % for T. equi, B. caballi, and mixed infections, respectively. The highest antibody levels against T. equi were detected in Kafr ElSheikh (40 %) and Giza (40.1 %) Governorates, whereas those of B. caballi were detected in Qalyubia (25 %) and Kafr ElSheikh (24.1 %) Governorates. Concerning T. equi, animals >10 years (OR = 2.06) were more likely to be infected with EP than those <5 years old. In addition, the seropositivity increased among grazing (OR = 5.7, 95 % CI: 1.73−19.27) males (OR = 1.8, 95 % CI: 1.23−2.61) infested with ticks (OR = 2.3, 95 % CI: 1.60−3.48) during summer (OR = 4.3, 95 %CI: 2.53−7.46); whereas the seropositivity of animals for B. caballi increased among grazing equines (OR = 7.8, 95 % CI: 1.05−58.25) over 10 years old (OR = 2.08, 95 % CI: 1.10−3.94) and infested with ticks (OR = 2.4, 95 % CI: 1.54−3.76) during summer (OR = 7.12, 95 % CI: 3.15–16.06). Therefore, EP is an important prevalent disease in Egypt and deserves further attention regarding the management system, treatment, and vector control.     

Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa

A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; ∼1600 bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.     

Control of equine piroplasmosis in Brazil.

The importance of equine piroplasmosis control in endemic countries has increased in recent years and plays an important role to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Babesia equi or Theileria equi and Babesia caballi) in Brazil--a country where the disease occurs endemically--in different climatic conditions, and to evaluate the results of a strategy for tick control in order to decrease infection rates. Blood samples were taken from 720 horses on 28 farms from different regions and subjected for complement fixation testing. The strategy was based on the control of the tick population by spraying the horses with acaricides, treating positive horses and preventing cattle and horses from grazing together. A significant association was found in the prevalence of antibody titres in tropical and subtropical areas. A significantly lower prevalence rate occurred on those farms where measures to control tick population were established. Farms in endemic countries may significantly reduce the prevalence of equine piroplasmosis by establishing measures to control the tick population and treating chronic carrier horses. Additional measures for controlling ticks in tropical areas are also discussed.     

Piroplasmosis in Donkeys—A Hematological and Serological Study in Central Ethiopia

Working donkeys (n = 395) originating from three different districts of the Oromia region of Ethiopia were examined October 2009 and April 2010 for the prevalence of equine piroplasmosis between. Apart from hematological and serological examination, tick vectors were collected and determined. Intraerythrocytic stages of Theileria equi and Babesia caballi were detected in 12.2% and 1.8% of examined stained blood smears, respectively. The seroprevalence of T. equi and B. caballi was 55.7% and 13.2%, respectively. The majority of active infection coincided with anemia. Only 16 donkeys were infested with four species of hard ticks: Rhipicephalus turanicus, Rhipicephalus evertsi evertsi, Rhipicephalus pulchellus, and Amblyomma variegatum.     

Prevalence and Significance of Parasites of Horses in Some States of Northern Nigeria

This study was conducted to determine the prevalence and significance of parasites of horses in northern Nigeria. Blood and faecal samples were randomly collected from 243 horses from different stables in some states of northern Nigeria for laboratory analyses. Fifty-seven horses (23.5%) were found infected with parasites. The hemoparasites detected, 21 (8.6%), include Theileria equi, Babesia caballi, Trypanosoma vivax and Trypanosoma evansi. The endoparasites encountered, 29 (11.9%) were Strongylus spp., Strongyloides spp., Oxyuris equi, Parascaris equorum, Paragonimus spp. and Dicrocoelium spp., 3 (1.2%) was Eimeria spp. Four horses (1.6%) had mixed infection of hemo- and endoparasites. This preliminary finding shows that parasitism is a problem in the horse stables examined, and calls for proper stable hygiene, routine tick control and regular deworming programme.     

Serological and molecular detection of Theileria equi in sport horses of northeastern Brazil

Theileriosis is a worldwide protozoal tick-borne disease caused by Theileria equi, which may produce a variety of clinical signs and turn infected horses into lifetime carriers. This study has aimed to perform a serological and molecular detection of T. equi and associated factors in sports horses from six areas of northeastern Brazil. In overall, 59.6% horses were positive by indirect immunofluorescence assay and 50.4% by polymerase chain reaction. No significant association was found when presence of ticks, age, gender, anemia or total plasma proteins was analyzed with seropositivity and molecular techniques. Although a significant association of infection was found in two cities. Thus, local risk factors other than presence of ticks, horse age, gender, anemia and total plasmatic proteins may dictate prevalence of T. equi infection in sports horses, even in highly endemic areas with no control of infection prior to horse competitions.     

Prevalence of piroplasmosis in equines in the Colombian Province of Cordoba

Summary Eighty-two equine sera from 13 farms in northern Colombia were examined for antibodies toBabesia caballi andB. equi using the complement fixation (CF) and the indirect fluorescent antibody (IFA) test. Seroreactors to both piroplasms were present on all farms. The IFA test indicated a prevalence of 90% forB. caballi and 94% forB. equi. The CF test detected antibodies toB. caballi in 41% and toB. equi in 65% of the animals. The prevalence of seroreactors in different age groups revealed a significant decline in CF antibodies toB. caballi in animals older than three years. IFA titres for bothBabesia spp. gradually declined with increasing age of the animals but were still present in most animals of the oldest age group (over nine years old).Anocentor nitens was found on all farms whereasAmblyomma cajennense was found only on two farms located on the coast.

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Prevalencia De Piroplasmosis En Equinos En La Provincia Colombiana De Cordoba

Resumen Se examinaron 82 sueros equinos provenientes de 13 fincas en el norte de Colombia, por la presencia de anticuerpos deBabesia caballi yB. equi, mediante la fijación del complemento y la prueba indirecta de anticuerpos fluorescentes. Se encontraron seroreactores a ambos piroplasmas en todas las fincas. La prueba de anticuerpos fluorescentes reveló una prevalencia de 90% paraB. caballi y 94% paraB. equi. La prueba de fijación del complemento detectó anticuerpos deB. caballi en 41% de animales y deB. equi en 65%. La prevalencia de seroreactores en grupos de diferentes edades reveló un descenso significativo en anticuerpos fijadores del complemento deB. caballi en animales mayores de tres años. Los títulos de anticuerpos fluorescentes para ambos piroplasmas declinaron con la edad, pero siempre estuvieron presentes inclusive en animales de nueve años de edad y más. La garrapataAnocentor nitens se encontró en todas las fincas, y elAmblyomma cajennense se encontró únicamente en dos de las fincas estudiadas.

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Prevalence De La Piroplasmose Equine Dans La Province De Cordoba, Colombie

Résumé On a recherché sur 82 sérums de chevaux de 13 fermes de Colombie du nord les anticorps anti-Babesia caballi etB. equi par les tests de fixation du complément (FC) et d'immunofluorescence indirecte (IFA). On a trouvé des sérums positifs dans toutes les fermes. Le test IFA donne une prévalence de 90% pourB. caballi et 94% pourB. equi. Le test FC détecte des anticorps anti-B. caballi chez 41% et anti-B. equi chez 65% des animaux. La prévalence des réagissants dans les groupes d'âge montre un déclin significatif des anticorps fixant le complément pourB. caballi chez les animaux de plus de 3 ans. Les titres IFA pour les deuxBabesia spp. déclinent graduellement avec l'âge mais sont toujours présents chez la plupart des animaux du groupe le plus âgé (plus de 9 ans). On a trouvéAnocentor nitens dans toutes les fermes, maisAmblyomna cajennense dans uniquement deux fermes de la région côtière.

     

Prevalence of the causative agents of equine piroplasmosis in the South West of The Netherlands and the identification of two autochthonous clinical Theileria equi infections

Equine piroplasmosis (EP) has not been considered indigenous in The Netherlands. However, following the detection of an apparently indigenous subclinical Babesia caballi infection in a horse on Schouwen-Duiveland (an island in the Zeeland Province), a survey was undertaken between May and September 2010 to assess the prevalence of the causative agents of EP in the South-West of The Netherlands. Blood samples from 300 randomly selected horses were tested for specific antibodies against Theileria equi and B. caballi using an indirect fluorescence antibody test (IFAT), and for parasite DNA using a specific polymerase chain reaction combined with reverse line blotting (PCR-RLB). Twelve of the horses (4%) were seropositive for EP. Of these, nine (75%) were positive (titre?1:160) for B. caballi alone and three (25%) were also positive for T. equi. PCR-RLB detected T. equi DNA in five horses (1.6%), two of which were seronegative. Four (1.3%) of the positive horses (three positive for T. equi and one for both B. caballi and T. equi) were considered truly indigenous. During the study, two indigenous ponies from a farm situated outside the sampling area were diagnosed with acute clinical piroplasmosis characterized by severe anaemia and pyrexia. Blood smears showed T. equi - like inclusions in red blood cells, and T. equi infection was confirmed in both ponies by PCR-RLB. The initial subclinical B. caballi infection, the survey results and the two acute clinical EP cases confirmed the autochthonous transmission of B. caballi and T. equi infections in The Netherlands.     

Comparative serologic study of equine piroplasmosis, with card and complement-fixation tests.

An agglutinating antigen and a rapid card test (CT) for equine piroplasmosis was developed. The antigen for the CT was prepared from lyophilized Babesia caballi complement-fixation (CF) antigen. Serum and plasma samples for testing were obtained from known B caballi-infected horses and clinically normal horses maintained at the laboratory. Serum samples also were obtained from horses outside the continental United States, in areas where piroplasmosis is endemic. Comparative CT and CF tests were done on all samples. The CT correctly identified 85% of 192 plasma samples from known infected and normal horses and 92% of 188 serum samples from these same horses. The CT results agreed closely with CF results. There was good agreement between CT and CF results. There was good agreement between CT and CF results on serum samples from horses outside the United States. Of 19 CF-positive samples, 90% were also CT-positive and 92% of 177 CF-negative samples were also CT-negative.     

Septic funiculitis caused by Streptococcus equi subspecies equi infection with associated immune‐mediated haemolytic anaemia

A 2‐year‐old Quarter Horse gelding presented for anaemia, icterus, depression and intermittent colic 2 weeks after routine castration. Bilateral septic funiculitis with Streptoccocus equi ssp. equi with secondary immune‐mediated haemolytic anaemia were diagnosed. A blood transfusion was required to facilitate general anaesthesia for surgical excision of the septic funiculitis. Antibiotic therapy was provided initially with chloramphenicol and later enrofloxacin. Immunosuppressive therapy was provided with dexamethasone and later azathioprine. The horse responded well to treatment and was discharged 8 weeks after presentation. Streptococcus equi ssp. equi should be considered in cases with septic funiculitis and the potential for a secondary immune‐mediated haemolytic anaemia exists with this bacterial species.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.