Theileria equi in the horses of Iran: Molecular detection, genetic diversity, and hematological findings

Highlights

• T. equi and B. caballi prevalence and diversity in horses of Iran were determined.

• Hematological alterations of piroplasma infected horses were analyzed.

• Prevalence of T. equi but not B. caballi was more than national average prevalence.

• PCR positive horses showed anemia, thrombocytopenia, neutrophilia, and monocytosis.

• EMA-1 phylogram showed a considerable diversity in Iranian T. equi strains.

Abstract

In all equids worldwide, Theileria equi and Babesia caballi are believed to be two important erythrocytic protozoa that cause equine piroplasmosis. In addition, it was recently discovered that Theileria haneyi is another potential equine piroplasmosis (EP) agent. Ixodid ticks are the major vectors of these parasites. Equine piroplasmosis is of international importance and affects enormously the equine industry. In this study, for the first time, molecular prevalence and genetic diversity of piroplasma parasites (T. equi and B. caballi) in horses from Fars province (south of Iran) were determined. Also, hematological alterations of naturally infected horses were analyzed. PCR positive horses showed anemia, thrombocytopenia, leukocytosis with a left shift of neutrophilia, and monocytosis. PCR results revealed that, from 133 blood samples of horses, 40 samples were positive (30.07%). The occurrence of T. equi in this area (30.07%) was more than the national average prevalence of T. equi (24.11%), but B. caballi prevalence in study area (0%) was less than the average of previous studies in Iran (5.47%). Our findings revealed that the T. equi was widespread in Fars province of Iran. PCR products of 18S rDNA and EMA-1 genes of T. equi strains were sequenced successfully. All 18S rDNA sequences collected in this experiment revealed 100% similarity together. According to the phylogenetic tree constructed using the 18S rDNA gene, Iranian T. equi is clustered with strains from Cuba (KY111762, KY111761) and USA (CP001669, JX177672). So, this could be concluded that T. equi studied in this research, and those strains are initiated from a common T. equi ancestor at an unknown time ago. Also, the phylogenetic tree based on EMA-1 gene demonstrated a genetically diverse population of Iranian T. equi strains (10 different genotypes). As EMA-1 is one of the most immunogenic antigens in this parasite, such variability could be a concern about the efficacy of T. equi vaccines. Finally, more studies on equine piroplasmosis in the provinces of the southern region of Iran are recommended to create a better vision of disease in this region.

Introduction

Two common erythrocyte organisms, Theileria equi and Babesia caballi, and a newly identified North American species, Theileria haneyi, cause a parasitic disease called equine piroplasmosis (EP). (Knowles et al., 2018; Tamzali, 2013; Wise et al., 2013). Equine piroplasmosis affects all equids, including horses, donkeys, mules, and zebras. The disease is considered an illness transmitted by ticks that leads to acute anemia (Mahmoud et al., 2016). A wide variety of ixodid ticks, including Amblyoma, Hyalomma, and Rhipicephalus are the principal vectors of EP (Scoles and Ueti, 2015). But there have been no investigations regarding vector potential for T. haneyi (Idoko et al., 2021). Besides, other transmission routes are also possible such as iatrogenic or mechanical transfer by infected needles and syringe, blood transfusion, surgical tools. Additionally, there is evidence of trans-placental transfer from pregnant mares to fetus, which was mostly leading to miscarriage (De Waal and van Heerden, 2004). Infected animals are long-term reservoirs of these blood parasites and function as a source of dissemination for the competent arthropods (ticks), which transfer the agents to host equines (Sumbria et al., 2014). Although animals recovered from T. equi infection will remain carriers lifelong, it takes four years infected animals become free of B. caballi, in a self-limited manner (Rothschild, 2013). In tropical and subtropical areas where tick vectors live, EP is endemic, and its distribution is worldwide, recorded in Asia, Africa, South and Central America, southern Europe, and some zones of the south USA (Salim et al., 2013). The lately described T. haneyi has been reported from several parts of the world, such as North America (Knowles et al., 2018; Sears et al., 2019), South Africa (Bhoora et al., 2020), Egypt (Elsawy et al., 2021), and Nigeria (Idoko et al., 2021; Mshelia et al., 2020). However, it has not yet been reported in the Middle East region. EP infection is of international importance, and adversely affects the equine industry, i. e. infected horses cannot pass through boundaries to participate in races and shows, and also their use for breeding aims and being sold is prohibited by the law (Brüning, 1996; Guidi et al., 2015).

Reduced hemoglobin levels, platelets, and red blood cells are the most frequent changes of hematological indices, and the anemia caused by T. equi is substantially more severe than B. caballi (Camacho et al., 2005). Horses infected with either T. equi or T. haneyi are reported to develop mild to moderate anemia, but the PCV index did not show any significant difference between them. However, the experimental study's clinical findings indicated that compared to other previously described isolates of T. equi, T. haneyi might be less virulent. (Sears et al., 2019).

Ergo, the principal aims of this experiment are as follows: i) Examine the existence of T. equi and B. caballi, and determine its prevalence in horses from south of Iran ii) Investigation of genetic diversity of piroplasmosis etiological agents in horses, and iii) Analysis of hematological alterations in horses naturally infected with EP.