A Survey Study on Sheep Theileriosis in Al-Fallujah City, Iraq

Theileriosis is considered one of the most important protozoan diseases affecting animals in tropical and subtropical regions of the world (Irfan et al., 2023; Ali et al., 2024; Mustafa et al., 2024). In sheep, the disease is primarily caused by Theileria ovis and Theileria herci, which are transmitted by various tick species, including Hyalomma spp. and Haemaphysalis spp. (Mans et al., 2015). Infection with Theileria can lead to a range of clinical signs such as fever, coughing, lethargy, lymphadenopathy, emaciation, and in severe cases, death (Ali et al., 2020; El-Imam and Taha, 2015).

Theileria species can be identified through microscopic examination of blood and lymph node smears (Faraj and Assi, 2022). Several serological assays have been developed to detect recent infections, including the indirect fluorescent antibody test (IFAT), complement fixation test (CFT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA), which has been widely used in seroepidemiological surveys of bovine theileriosis. These assays often target specific antigens such as the Theileria luwenshuni surface protein. Notably, the iELISA method demonstrated high diagnostic performance, with a reported specificity of 98.9% and sensitivity of 98.5% (Salman et al., 2014; Li et al., 2016).

In addition to serological methods, Theileria infection can also be detected using molecular techniques such as polymerase chain reaction (PCR) (Salih et al., 2024). However, traditional PCR methods may not effectively differentiate between clinically infected animals and carrier animals, as they do not quantify parasite load (Faraj and Assi, 2022). To address this limitation, quantitative PCR (qPCR) has been employed for more accurate diagnosis and quantification of Theileria species (Selim et al., 2021).

The present study aimed to detect Theileria infection in sheep using the ELISA method and to investigate the effects of location, month, age, and gender on infection rates.

Ninety blood samples were randomly taken from sheep aged less than one to four years, 30 males and 60 females, from different areas of Fallujah, Al-Shahabi, Al-Saqlawiyah, and Al-Karmah, for the period from November 2024 to March 2025 (Hussein et al., 2024).

For serological studies, 5 ml of jugular vein blood was collected after cleaning and disinfecting the area with (70%) ethyl alcohol. The blood was collected into sterile, anticoagulant-free tubes. Then allowed to clot, and the serum was isolated by centrifugation at 3,000 g/ 10 minutes. The separated serum was stored in 1.5 ml Eppendorf bottles at -20°C until use.

An ELISA test kit for ovine Theileria (SunLong Biotechnology Co., Ltd., China) was used to detect antibodies in the serum samples. The procedure involved the standard ELISA steps: sample addition, incubation, washing, staining, and reaction termination. Serum samples were mixed with a diluent and sample buffer according to the manufacturer’s instructions. After processing, the microplate was read using a microplate reader, and optical density (OD) values were recorded. The critical (cut-off) value was calculated based on the method described by Gao et al. (2002), following the protocols established for the assay.

A Chi-square test was used to analyze all data and compare the different factors affecting the infection rate. A P value of ≤ 0.05 was considered statistically significant (Kwak, 2023).

According to Table 1, the overall infection rate with theileriosis in this study was 30%. This is comparable to infection rates reported in other studies, such as 33.8% by Aktas et al. (2005) and 22.8% by Al-Amri and Hasso (2002). The current prevalence is higher than the 7.5% reported in Mosul Governorate (Al-Fatly, 2012), but lower than the 52.4% recorded by Hamid and Al-Obaidi (2023). Similar variations have been observed in neighboring countries; for instance, Iran and the Sultanate of Oman reported infection rates of 11.9% and 17%, respectively (Dhim and Aaiz, 2014; Razmi et al., 2006). In Sudan, a slightly higher infection rate of 22.1% was documented (Muslih et al., 1988), whereas Egypt reported a much higher prevalence of 77.93% (Ahmed et al., 2003). These differences may be attributed to factors such as variations in sheep breeds, environmental conditions, the immune status of the animals, sample size, and vector density.

Table 1: Incidence of Theileria infection in sheep as detected by ELISA.

Our study revealed significant heterogeneity (P ≤ 0.05) in infection rates across different study areas. The highest prevalence was observed in Al-Saqlawiyah (50%), while the lowest was in Al-Fallujah Centre (16.6%) as shown in Table 2. These significant differences (P ≤ 0.05) could be related to variations in the distribution of vector ticks (hard ticks) across these regions, differences in immunity acquired from previous infections, or disparities in the regular use of treatments between areas. Tageldin et al. (2005) similarly reported that infection rates varied across different regions of Pakistan, largely due to differences in climate.

The highest prevalence of Theileria infection was observed in sheep older than 4 years (35%), followed by those aged 1–4 years (30%), while the lowest prevalence was recorded in the age group under 1 year (20%), with a significant difference (P ≤ 0.05) (Table 3). This finding contrasts with that of Fadel et al. (2010), who reported a higher prevalence in lambs (78–85%) and a lower prevalence (9%) in adult animals. However, it aligns with Mohsin et al. (2022), who documented a low infection rate in lambs under six months of age. Similarly, Luo and Yin (1997) observed a greater incidence of infection in sheep older than three years. The lower infection rate in lambs may be attributed to the passive transfer of immunity from their dams (Naz et al., 2012) or the protective effects of treatments administered to young animals (Khalaf, 2009).

Table 2: Prevalence of Theileria infection in sheep according to study areas.

Table 3: Age-related frequency of Theileria infection in sheep.

Regarding monthly prevalence, the highest infection rate was recorded in November (52%), followed by March (35.3%) and December (30%). February showed a lower infection rate of 20%, while no infections were detected in January. These differences were statistically significant (P ≤ 0.05) (Table 4). This finding contradicts the observations of Mohsin et al. (2022) and Ahmed et al. (2008), who reported no significant variation in Theileria infection rates across different months. The discrepancy in monthly infection rates in this study may be attributed to seasonal variations in temperature and humidity, which influence tick infestation levels (Salih, et al., 2024).

The current investigation revealed a significant difference (P ≤ 0.05) in Theileria infection prevalence between females and males. Females showed a higher prevalence (31.6%) compared to males (26.6%), as presented in Table 5. This statistically significant association (P ≤ 0.05) between gender and infection may be attributed to stress factors related to milk production and gestation in females (Zahid et al., 2005). Additionally, females may have a longer lifespan in the population since males are often sent to slaughter earlier. In contrast, Mohsin et al. (2022) reported no significant effect of gender on infection rates, which they attributed to equal exposure of both sexes to the same environmental conditions and vectors.

Table 4: Monthly distribution of Theileria infection in sheep.

Table 5: Gender-wise prevalence of Theileria infection in sheep.

The prevalence of theileriosis in sheep across different areas of the region was 30%, with significant variations observed among study locations, months, age groups, and sexes. This study provides the first scientific documentation of a high prevalence of theileriosis in sheep within these specific areas. Additionally, the findings highlight competitive ELISA as a reliable diagnostic tool for detecting infection in the vector host, offering valuable potential for improving epidemiological surveillance and informing control strategies in the future.

We gratefully acknowledge the Deanship of the Faculty of Veterinary Medicine at the University of Fallujah for helping and supporting this project.

This study is the first to decomented a survey of the parasite Theileria spp. In sheep from Fallujah city, Iraq through ELIZA techneq. The results highlight disease conditions and the influence of age, gender, and months, in addition to the different rural areas where animals are found.

All authors contributed to the manuscript equally in collecting blood samples, analyzing them using ELISA, organizing data, and academic writing.

The study was approved by the Council of the College of Veterinary Medicine, University of Fallujah, under approval number [8], dated 25 May 2025.

The authors have declared no conflict of interest