PhD Thesis defense by Eliazar Camino Gutiérrez at the VISAVET Centre of the Complutense University of Madrid

July 13^rd, 2021

Equine piroplasmosis (EP) is a parasitic disease of equids (horses, donkeys, mules and zebras) caused by two intraerythrocytic protozoan parasites within the phylum Apicomplexa: Theileria equi (previously named Babesia equi) and Babesia caballi. These parasites are mainly trasmitted by ixodid tick species belonging to several genera such as Dermacentor, Hyalomma and Rhipicephalus; however, transmission may also occur vertically in utero and iatrogenically. This disease has a hyperacute, acute, subacute or chronic form; affected animals often show variable and nonspecific clinical signs such as: fever (often exceeding 40ºC), pale mucous membranes, jaundice, peripheral edema, anorexia, weight loss, decreased performance and splenomegaly. Horses can remain carriers of these parasites for a long time after the resolution of acute disease, being a source of infection for ticks and other horses.

EP is endemic in many southern European countries (including Spain), Africa, Asia, Central and South America. Countries such as United States, Canada, Japan, New Zealand and Australia are “EP-free”, a status maintained by restrictions on importation of EP seropositive horses. This requirement limits the international movement of horses, causing severe economic losses to the Spanish equine sector, since a large number of horses test seropositive for EP. This fact especially affects the trade of Spanish Purebred (SP) horses, which account around 85% of purebred horses in Spain and almost 100% of exports to Europe and America.

To date, EP has only been studied in certain geographic regions of our country and veterinary surgeons often base the diagnosis and treatment of this disease on clinical and epidemiological criteria, without having a confirmation by the laboratory of the presence of these parasites in the bloodstream. The main objectives of this doctoral thesis included the determination of the current epidemiological situation and genetic diversity of T. equi and B. caballi nationwide, the estimation of the economic losses that EP involves for the Spanish equine industry and the evaluation of the most appropriate diagnostic tests in horses with clinical suspicion of this disease. Five studies were performed: a pilot study in central Spain, a nationwide study and three complementary studies.

The pilot study was carried out by sampling at SP breeding stud farms located in central region of Spain. This study revealed the presence of at least one seropositive horse in 81.1% (68.2% -94.0%) of the stud farms together with the existence of a moderate exposure [24.9% (21.3% -28.6%)] of these horses to T equi and/or B. caballi, demonstrating central Spain can be considered as high-risk area for EP infection.

In the national study, samples of apparently healthy horses were collected using a random stratified sampling based on the horse census in each Spanish autonomous community. The high seroprevalence of EP [42.9% (39.4-46.5)] and the considerable percentage of asymptomatic carriers horses found [30.3% (27.0-33.6)] confirmed the endemic situation of this disease in our country, being the exposure to T. equi [35.8% (32.3- 39.25)] two-fold higher than B. caballi [15.6% (13.0-18.2)]. Navarre and Extremadura were the autonomous communities with the highest levels of positivity to EP, while the lowest levels were found in Castile La Mancha, Andalusia and in the eastern area of Spain.

Within the two previous studies, a questionnaire was designed to cover the main characteristics of each sampled horse and its farm with the aim to identifying risk factors associated with higher levels of EP seroprevalence. The number of stallions residing in the stud farms, the increase in horse age, the presence of ticks and the contact with cattle were factors related to a higher seropositivity to EP. By contrast, the implementation of vaccination programs and disinfection measures in the stud farms, together with fairs/competitions attendance were factors associated with the control of this disease.

The complementary study 1 included blood samples from horses showing clinical signs of EP. Hematological, biochemical, immunological and molecular analysis were carried out on these samples. Horses parasitized by B. caballi showed a more severe hemolytic anemia, whereas T. equi infections were mostly associated with leukocytosis. The level of agreement between EP results of serological and molecular tests was moderate (k = 0.587); however, blood smears examination revealed the presence of hemoprotozoan agents compatible with T. equi and/or B. caballi in only 9.3% of horses, even when 42.9% of horses tested EP positive by PCR. These results confirm PCR is the test of choice in order to confirm the presence of these parasites in clinical cases of EP. In addition, certain hematological parameters can be considered as biomarkers of the infection caused by one or another parasite.

The complementary study 2 was developed in order to have a greater knowledge of the economic losses that EP involves for the exportation of horses from Spain to EP-free countries. Serum samples from horses collected prior to export for a period of three years (2015-2016, 2016-2017 and 2017-2018) were tested by serological analysis. Serological results revealed that 24.1% (22.6-25.5) of horses could not be exported to these countries, meaning that 14.5 million euro would be lost each year in Spain. Moreover, there was a significant increase of 2.2% for the number of horses seropositive to B. caballi during this period (2015-2018).

Finally, the complementary study 3 investigated the presence and geographical distribution of T. equi and B. caballi genotypes circulating currently within the country. Phylogenetic analysis revealed that the T. equi isolates grouped into three genotypes (A, D and E), while B. caballi isolates were place into two genotypes (A and B). Isolates from T. equi genotype D and B. caballi genotype B had not previously been reported in Spain. Genotype A of B. caballi was only found in northern regions. However, T. equi showed a widespread distribution, locating genotype E in all sampled communities and genotype A in central and southern areas of Spain.

The results of this doctoral thesis indicate the existence of a high exposure to T. equi and B. caballi nationwide together with a possible spread of these parasites at present, a finding mainly associated with the uncontrolled movement of horses both between the different Spanish autonomous communities and between Spain and other EP-endemic countries. The implementation of an annual epidemiological surveillance programme and mandatory serological screening of horses prior to movements could aid to control this disease in our country.