INTRODUCTION

Blood parasites of cattle are microscopic pathogens transmitted primarily by ticks and cause substantial morbidity, mortality, and economic losses through reduced productivity, impaired animal health, and, in some cases, the emergence of drug-resistant strains [1, 2]. Among these pathogens, bovine theileriosis is one of the most important tick-borne diseases and is caused by several Theileria species, each exhibiting distinct host specificity, geographic distribution, and pathogenicity [3]. For example, Theileria_ annulata_ infects cattle and other susceptible hosts across tropical, subtropical, and Mediterranean regions, causing severe tropical theileriosis with high morbidity and mortality, whereas T. parva is predominantly distributed in sub-Saharan Africa and is responsible for the highly fatal East Coast fever in cattle [4, 5].

T. orientalis has a worldwide distribution and primarily infects cattle, typically causing a mild, non-transforming, and frequently asymptomatic infection. Nevertheless, the parasite has emerged as an important cause of bovine theileriosis because it can induce hemolytic anemia, reduced milk production, decreased weight gain, reproductive losses, and considerable economic damage to the cattle industry [6, 7]. The genus Theileria possesses a complex life cycle involving schizonts in lymphoid cells and piroplasms within erythrocytes of vertebrate hosts, whereas ticks become infected by ingesting parasitized erythrocytes during blood feeding [8, 9]. Within the tick gut, erythrocytes are lysed, releasing piroplasms that differentiate into gametes and subsequently fuse to form zygotes. These zygotes penetrate the gut epithelium, develop into kinetes, migrate to the salivary glands, and undergo sporogony to produce infective sporozoites. During subsequent blood feeding, sporozoites are transmitted through tick saliva into the mammalian host, where they invade leukocytes, develop into schizonts, and eventually produce erythrocytic piroplasms [10–12]. Although acute clinical signs such as pyrexia and anemia may be absent or transient, T. orientalis infections often persist throughout the host's lifetime, with recrudescence occurring during periods of physiological or environmental stress, including lactation, pregnancy, and sudden environmental changes [13, 14].

Accurate diagnosis of T. orientalis infection is essential for effective epidemiological surveillance and disease control. Conventional microscopic examination of Giemsa-stained blood smears has limited sensitivity and specificity, particularly in animals with low parasitemia or chronic infections. Consequently, molecular diagnostic methods, especially polymerase chain reaction (PCR), have become the preferred approach due to their superior sensitivity, specificity, and ability to detect multiple T. orientalis genotypes [15, 16]. Furthermore, DNA sequencing provides valuable information on genetic diversity, evolutionary relationships, and molecular epidemiology, enabling accurate characterization of circulating isolates and comparison with global reference strains [17, 18].

In Iraq, several investigations have documented the occurrence of different Theileria species in cattle and other domestic animals [19–22]. However, molecular information regarding Theileria infections in ticks remains extremely limited, with only a few published studies investigating tick-associated infections [23]. Moreover, molecular confirmation of T. orientalis in Iraqi cattle has been reported only once, from Fallujah City in Al-Anbar Province [24]. To date, no study has simultaneously investigated genetically related T. orientalis isolates in cattle and their associated ticks or examined their phylogenetic relationships with globally circulating strains. Consequently, important knowledge gaps remain regarding the molecular epidemiology, host-vector relationship, genetic diversity, and potential transmission dynamics of T. orientalis in Iraq.

Despite the increasing recognition of T. orientalis as an emerging tick-borne pathogen worldwide, comprehensive molecular epidemiological studies in Iraq remain scarce. Previous investigations have largely focused on detecting Theileria species in cattle, while molecular characterization of parasites circulating in naturally infesting ticks has received little attention. Furthermore, no previous Iraqi study has simultaneously evaluated cattle and corresponding tick isolates using sequence-based phylogenetic analysis to determine their genetic relatedness and relationship with international reference isolates. This lack of integrated host-vector molecular data limits the current understanding of transmission pathways, genetic diversity, and regional dissemination of T. orientalis, thereby hindering the development of evidence-based surveillance and control strategies.

Therefore, this study aimed to determine the molecular prevalence of T. orientalis in naturally infected cattle and their associated ticks from Wasit Province, Iraq, using PCR targeting the 18S rRNA gene. In addition, the study sought to characterize the genetic diversity of the detected isolates through DNA sequencing, evaluate their phylogenetic relationships with globally reported T. orientalis strains, and investigate host-associated risk factors influencing infection. Collectively, these findings provide the first integrated molecular and phylogenetic evidence linking cattle and tick isolates of T. orientalis in Wasit Province and establish an important baseline for future epidemiological surveillance and control of oriental theileriosis in Iraq.