Sheep industry is a significant economic asset due to the animals' resilience to food shortages and weather fluctuations (1). In Egypt, sheep and goats play a vital role in providing meat to humans, along with serving as sources for wool production and utilizing their manure as a fertilizer for soil (2). Nevertheless, sheep are vulnerable to numerous haemoprotozoan diseases that can impact their health and productivity. Among various others, Theileria spp. and Babesia spp., responsible for theileriosis and babesiosis, respectively, stand out as highly economically significant tick-borne diseases transmitted to sheep (3). These haemoprotozoa are part of the broader classification belonging to the Class Sporozoa, Order Piroplasmida, and families Babesiidae and Theileriidae (4). In terms of transmission, Theileria and Babesia are biologically transmitted by hard ticks in tropical and subtropical regions globally (5). Both diseases frequently occur between May and September, coinciding with the periodic movement of ticks (6). Regarding the life cycle of Babesia (7), the organism undergoes asexual division within a mammalian red blood cell (RBC), after which the daughter merozoites rupture the host cell to infect new erythrocytes. Ticks become infected by consuming erythrocytes containing merozoites. Sexual reproduction (gametogony) takes place in the tick gut, followed by asexual sporogony in its salivary glands. Subsequently, the development of infective stages (sporozoites) occurs in the salivary glands of infected ticks. This cycle can persist throughout the animal's lifespan depending on the host's immunity. Regarding their transmission, two distinct routes of transmission are involved: transstadial transmission from one stage of the tick's life cycle to the next and transovarial transmission (8–10). The life cycle of Theileria closely resembles that of Babesia, involving sexual gametogony followed by asexual sporogony in the tick gut. Ultimately, the inoculated sporozoites invade lymphocytes in the infected host, prompting continuous division as lymphoblasts through a process known as cellular transformation. This results in parasite replication, and the infected cells become widely distributed throughout the host's lymphoid system. After 2–3 weeks, cytotoxic T-cells damage parasitized lymphoblasts. Simultaneously, the parasites initiate an attack on erythrocytes, reproducing asexually within them. Ticks become infected through the ingestion of these infected erythrocytes. Transstadial transmission is the only established mode of transmission in Theileria spp., with no evidence of discernible transovarial transmission (11). Among others, Rhipicephalus (Boophilus) annulatus and other members genus Hyalomma, such as H. marginatum and H. excavatum, are considered the most predominant ticks responsible for transmittion of piroplasmosis to sheep in Egypt (12, 13).

Ovine babesiosis is a significant tick-borne disease affecting small ruminants, caused by various species such as B. ovis, B. motasi, B. crassa, B. taylori, and B. aktasi, among others (6, 7, 10–12, 14–16). Taken into account, the most pathogenic species in sheep is B. ovis that causes malignant ovine babesiosis with fever, hemolytic anemia, hemoglobinuria, icterus and possible death others (6, 7, 10–12, 17, 18). Hemoglobinuria is uncommon, but it can occur in the advanced stages of the disease, leading to potential abortion in pregnant animals. Herds at risk can experience a significant mortality rate (19). Sheep with chronic infections typically display no symptoms, except for the presence of parasitemia and a lack of thriftiness. Recovered animals with latent infections usually possess immunity for a specific period, and there is no cross-immunity between the parasites (20). Theileriosis in small ruminants is caused by protozoan parasites of the Theileria, specifically including species such as Theileria lestoquardi, T. ovis, T. recondita, T. annulata, T. separata, T. luwenshuni, T. uilenbergi, Theileria sp. OT1 and Theileria sp. OT3 (7, 21). Common symptoms of theileriosis in sheep include cough, fever, lymphadenopathy, fatigue, and weight loss (22). Among others, T. lestoquardi is considered the most pathogenic species, leading to a malignant ovine theileriosis with a great morbidity and mortality proportion in sheep (23). Also, several previous investigations revealed that Theileria infection can result in abortion (24, 25). The diagnosis of ovine babesiosis and theileriosis can be achieved through clinical symptoms observation and microscopic examination of lymph or thin blood smears stained with Giemsa which remains the gold standard for diagnosis of the infection. However, it should be noted that relying solely on morphological characteristics for blood parasite detection (26, 27). On the other hand, molecular identification of these parasites offers greater sensitivity and specificity compared to alternative diagnostic methods (15). However, the cost of these later techniques poses a significant challenge, particularly in countries with limited resources.

It should be stressed that blood parasites pose a significant economic challenge in sheep production in Egypt, leading to losses in meat and milk production due to high parasitemia and mortality rates in infected animals (28, 29). Clearly, routine surveillance of these parasites among small ruminants in a particular area is essential for understanding the prevalence of these diseases and implementing effective control measures. While some prior studies have been conducted on ovine babesiosis and theileriosis in Egypt [Table 1; references (4, 30–32, 34–39)], there is a notable scarcity of research, especially the molecular work in the southern region of the country, namely Upper Egypt. Considering the importance of understanding factors affecting infection exposure within natural populations, it is crucial for comprehending host-parasite dynamics, predicting infection susceptibility, and maintaining biological equilibrium (40). While several factors elucidated differences in occurrence of the parasite and exposure to infection among host species, the relationship between hosts, ticks, and pathogens undergoes continuous changes, largely driven by ecological, climatic, and anthropogenic alterations. Various factors, including animal sex, age, herd management practices, seasonal variations, tick infestation, and herd size, have been associated with infection by blood parasite in animals. Additionally, spatio-temporal factors such as vector habitats, animal feeding systems, sanitation measures, and management practices significantly influence the epidemiology of these infections (41). As mentioned above, theileriosis and babesiosis are parasitic diseases of significant commercial importance, thereby playing a crucial role in the global trade of animals and animal products. The precise documentation of these parasites in Egypt is essential for understanding their epidemiology and classification, enabling effective control measures to mitigate the damage caused by their infections. Given the aforementioned details, the present study aims to investigate the prevalence and epidemiology of ovine babesiosis and theileriosis in Assiut Governorate, Upper Egypt, while also considering the potential associated risk factors. This assessment utilizes a combination of microscopic methods, followed by molecular identification of the parasites.

Piroplasmid infection poses a significant threat to sheep populations globally. Considering their veterinary significance and substantial economic implications, understanding the epidemiological pattern of piroplasm's infection is essential for implementing targeted control strategies, including tick control measures, vaccination programs, and improved management practices to reduce the burden of the disease in sheep populations. The current study delves into the exposure and epidemiology of these parasites specifically among sheep populations from Upper Egypt, together with molecular characterization of both parasites. In addition, the study reviewed the prevalence data of Babesia spp. and Theileria spp. among sheep in Egypt (Table 1). As illustrated, the current study revealed that 176 out of the tested samples tested positive for piroplasmosis using microscopical detection methods, resulting in an overall prevalence of 44%. At national level, a previous study conducted by Hussein et al. (31) in Qena governorate, Upper Egypt, reported an overall prevalence of blood parasites at 38.46% which is slightly lower than our present findings. Conversely, another previous research (33) in the North Coast of Egypt showed a higher prevalence rate of blood parasites (84.80%), surpassing the present reported findings. Regarding the individual prevalence rates reported, the infection rates detected for Babesia spp. and Theileria spp. were 14% and 30%, respectively. A previous study conducted (4) in Behera governorate (Lower Egypt) reported prevalence rates of 17% for Babesia and 20% for Theileria, respectively. Another previous study (29) documented presence of Babesia spp., with prevalence rates of 23.7%, 23.1%, 21.2%, and 20.6% in the governorates of Beni Suef, Kafr Elsheikh, El-Fayoum, and El-Sharkia, respectively, which surpasses the prevalence rates observed in the current study. Conversely, another study conducted on sheep from Kalubyia governorate (Lower Egypt) revealed a lower incidence of 8.5% for Babesia spp. (28). A previous research (56) conducted on sheep from Egypt reported a higher prevalence rate of 50.7% for Theileria spp. by Giemsa stained blood film (24–26, 38, 46, 48, 49). Variations in infection prevalence rates may stem from the intricate interactions of factors such as the presence of infected ticks, sanitary and hygienic practices, procedural techniques, immunological status of infected animals, and the absence of veterinary supervision (34, 57, 58). Abiotic factors, particularly temperature and humidity, have a significant impact on the epidemiology of these infections (59). The climatic conditions in the studied area are characterized by mean temperatures throughout the year ranging from 18°C to 40°C, establishing an optimal habitat for the enduring presence of hard ticks. This environment not only facilitates the proliferation of their hosts and the spread of associated blood parasites but also likely contributes to the elevated prevalence rates (60, 61). Concerning the reported clinical signs, the infected sheep exhibited symptoms such as anorexia, anemia, and emaciation. Additionally, hemoglobinuria recorded in sheep infected with Babesia spp. Those infected with Theileria spp. displayed additional clinical signs such as lacrimation, corneal opacity, and enlarged lymph nodes, consistent with observations reported in the literature (22).

Concerning age, which was explored as a potential risk factor associated with the exposure to those parasites, this study unveiled that the overall prevalence of blood parasites in sheep was non-significantly higher in young animals under 2 years, compared to adult ones over 2 years. In this concern, the study found that Babesia spp. infection rates were higher in animals under 2 years compared to those over 2 years, while Theileria spp. infection rates were also more prevalent in the younger age group. However, the statistical difference were not significant. In a prior study by Fadly, 2012 (4) in Behera, Upper Egypt, a higher prevalence of both Babesia spp. and Theileria spp. was higher in sheep above 3 years of age, which contrasts with our findings. Furthermore, Elsayed et al. (33) in North Coast of Egypt concluded that the infection with blood parasites was higher in sheep more than 3 years. Another study (62) in Egypt recorded a higher infection rate in animals aged ≤ 1 year. The discrepancies observed between the current findings and other studies could be attributed to variations in sanitary and hygienic practices, the number of examined animals, variations in immunological resistance, and the impact of local climatic conditions, which may influence the spread of ticks (32).

Regarding sex as a studied individual variable factor, this study found that the overall prevalence of blood parasites in male sheep was significantly higher compared to females, which aligns with earlier studies conducted nationally or internationally (63–66). However, the present findings are in contrast with another reports in Egypt (31, 33, 62). This variation may stem from a notable difference in the sample size of males and females included in our study which could influence the prevalence rates (67). Another contributing factor is management practices in Egypt, where farms typically have few breeding males but a high percentage of young male lambs (68) and the increased susceptibility to infection among females grazing together outdoors (31). It has been demonstrated that the contact of healthy livestock with infected livestock during free grazing is a critical risk factor for increasing the burden and spread of Theileria infection (69–71). Furthermore, other previous reports revealed higher occurrence of blood parasites in female as compared to males due to various physiological factors such as pregnancy, parturition, and milk production, may experience increased stress, rendering them more susceptible to infection than males (72). In addition, farm management, microclimatic patterns, tick distribution, host breed, and sampling conditions may account for the variation in prevalence rates of tick-borne pathogens (73, 74). In this study, the prevalence of blood parasites was significantly higher in the cold season 66.1% than in the hot season 15.9%. The infection rate of Theileria spp. and Babesia spp. was higher in cold season (44.6% and 21.4%, respectively) than in hot season (11.4% and 4.5% respectively). Similarly, another previous study detected higher prevalence of blood parasites in winter when compared to summer (63). The obtained result disagreed with those data recorded (4) in Behera governorate (Lower Egypt), who reported higher infection rate in summer months as compared to winter season. Other studies have documented that there were no differences in the prevalence rate of blood parasites across the analyzed seasons (36). This may be attributed to the presence of ticks and can be explained by the hypothesis that the average temperatures of the studied area during cold months, which are known to provide favorable conditions for the rapid proliferation of ticks and facilitate their easy transmission among susceptible animals (75). These variations can be attributed to geographic and climatic conditions, vector activity, the parasitic status of the investigated area, and the immunological status of the examined animals (4, 76). In this regard, the studied area provides a conducive environment for the development of hard ticks (39). Specifically, the average temperatures in this region during winter might reach 21°C (77, 78), providing optimal conditions for the year-round spread of host vectors and accompanying blood parasites (60, 61).

Regarding the phylogenetic analysis, this study unequivocally identified two bloods parasites species, B. ovis through 18S rRNA gene and T. annulata by tams1 gene. According to previous studies, a conserved gene of 18S rRNA is a specific target for Babesia and Theileria species detection by using PCR assay (12, 79, 80), being a sensitive diagnostic tool for ovine piroplasmids. On the other hand, the Tams1 gene has proven to be a tool to detect new related Theileria species (81). Concerning Theileria, while blood smear examination remains the primary method for diagnosing, it falls short in species differentiation due to morphological similarities (82). In this work, PCR technique allowed us to identify T. annulata in this study. Similarly, a previous work (32) in Egypt detected various Theilaria species (T. ovis, T. annulata and T. lestoquardi). Experimental infections of sheep and goats with T. annulata, the causative agent of tropical theileriosis in cattle, have been documented to induce mild symptoms without piroplasm development. However, previous findings suggest that T. annulata can cause mild clinical symptoms in sheep post-experimental infection, and T. lestoquardi can similarly affect cattle (83). Precisely, T. lestoquardi is considered the primary pathogenic species responsible for malignant ovine theileriosis (84, 85), with many positive samples likely attributed to this species taking into account its mix detection with other species in different studies (9, 76, 86). Nonetheless, another study (87) also identified natural infections of sheep with various species of Theileria, known as T. lestoquardi, T. ovis, and T. annulate, by nested PCR-RFLP. Both infections are transmitted by the same vector, H. anatolicum, which is found in Egypt (76) and in many areas, cattle and sheep are raised together, potentially leading to cross-infestation (88, 89). Due to logistical constraints, our study was unable to conduct molecular determinations on all samples. The findings reported here raise the possibility of sheep acting as reservoirs for T. annulata, provided both parasites and a competent vector coexist in the same area. Despite this challenge, this study is considered one of the few molecular reports in Egypt confirming B. ovis, although confirmation could not be achieved in all samples, highlighting the need for further studies in this area to accurately determine the prevalence of this species in Egypt.

The current study faces several methodological limitations that necessitate careful consideration when interpreting the findings. Initially, screening for both blood parasites involved examining only a single blood film sample per animal using microscopy. While this method remains the gold standard technique for large-scale screening of piroplasmosis, particularly in resource-constrained settings, its accuracy in detection may be compromised, potentially leading to an underestimation of the true infection rate. Secondly, the cross-sectional design of the study is inadequate for monitoring the progression of piroplasmosis and for capturing seasonal variations in infection rates. Thirdly, the findings obtained may not be generalizable to other epidemiological contexts or geographic regions within the same country. Finally, the relatively limited number of samples subjected to molecular characterization may have impacted the precision of estimating the true genetic diversity and frequency of parasite species circulating among the examined sheep.

The current study has provided significant insights into the prevalence and impact of Babesia and Theileria, reaffirming their ongoing importance as blood parasites affecting Egyptian sheep in upper Egypt. This investigation emphasizes the critical role of utilizing both traditional microscopy and cutting-edge molecular methods for the precise detection and characterization of piroplasmosis. Given the above-mentioned findings, of particular note is the apparent rise in the circulation of piroplasmosis, a trend likely worsened by the expanding populations of the tick vectors. Furthermore, the study sheds light on the current epidemiological landscape of piroplasmosis in the Egyptian environment. By addressing the spread of these parasites, informed actions can be taken to mitigate the associated diseases, safeguarding the health and wellbeing of both livestock and the population at large. These findings serve as a call to action, emphasizing the importance of ongoing surveillance and strategic interventions to manage and minimize the impact of Babesia and Theileria infections in Egypt. This underscores the urgent necessity for additional investigations centered on these vectors, signaling a critical domain for future research endeavors and intervention initiatives. Further studies with larger sample sizes, diverse small ruminants, and additional epidemiological data are also necessary to comprehensively evaluate the associated risk factors for infection. Such knowledge would be helpful for governmental authorities to develop and implement effective control measures.

The original contributions presented in the study are publicly available. This data can be found at: https://www.ncbi.nlm.nih.gov/nuccore/; OP984151.1.

The animal studies were approved by Research Ethics Committee of the Faculty of Veterinary Medicine, Assiut University, under the designation 06/2023/0130. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

AD: Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. FA-A: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing—original draft, Writing—review & editing. SM: Conceptualization, Methodology, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. AG: Conceptualization, Data curation, Formal analysis, Investigation, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. FO: Conceptualization, Data curation, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing. FE: Data curation, Formal analysis, Investigation, Software, Validation, Writing—review & editing. EH: Data curation, Formal analysis, Software, Validation, Visualization, Writing—review & editing. NA: Conceptualization, Data curation, Formal analysis, Software, Validation, Writing—review & editing. HA: Data curation, Formal analysis, Funding acquisition, Investigation, Software, Validation, Writing—review & editing. AA: Data curation, Formal analysis, Software, Validation, Writing—review & editing. DB-B: Data curation, Formal analysis, Software, Writing—review & editing. EE: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Validation, Writing—original draft, Writing—review & editing.