Infestation with the larvae of the sheep nasal bot fly, Oestrus ovis (Diptera: Oestridae), is widely recognized as a significant parasitic condition affecting small ruminants in diverse agro-ecological regions (Ahaduzzaman, 2019). The infection is commonly associated with respiratory manifestations such as persistent sneezing and serous to purulent nasal discharge, which can compromise animal welfare and productivity (Hanan, 2013). From an economic perspective, oestrosis may adversely affect animal performance, as the irritation and respiratory disturbances associated with O. ovis infestation can interfere with normal feeding behavior (Dorchies et al., 1998). These impacts are particularly critical in regions where small ruminants represent a major source of income and food security (Tella and Chineke, 2022).
       
The pathogenicity of O. ovis is primarily linked to the development of its larval stages within the nasal cavities and paranasal sinuses of the host, with occasional extension into deeper cranial structures (Hidalgo et al., 2015). Progressive accumulation of mucopurulent exudate, often mixed with environmental contaminants, may obstruct the upper respiratory tract and induce mouth breathing, thereby disrupting normal grazing and rumination behavior. Although the usual manifestations are primarily associated with irritation and inflammation of the upper respiratory tract, more severe complications have occasionally been described in heavily affected animals. Rare reports have also described aberrant larval migration and neurological involvement; however, such complications are uncommon and were not evaluated as outcomes in the present study (Dorchies et al., 2000; Alcaide et al., 2005; Karatepe et al., 2014; Ozdal et al., 2016).
       
Epidemiological studies have demonstrated that the prevalence and occurrence of O. ovis infestation are strongly influenced by environmental and climatic conditions (Cepeda-Palacios et al., 2011). In Saudi Arabia, relatively high infestation rates have been reported, with prevalence reaching 53.54% in the Jazan region (Hanan, 2013), indicating that local conditions are highly conducive to parasite transmission. The adult female fly is larviparous, depositing first-instar larvae directly into the nostrils of the host, where they undergo three successive developmental stages before exiting to pupate in the soil (Dincer, 1997; Kettle, 1995). The duration and dynamics of this life cycle vary according to seasonal patterns, which in turn affect transmission during different periods of the year.
       
In addition to its veterinary importance, O. ovis has zoonotic implications. Although relatively uncommon, human cases, particularly ophthalmomyiasis, have been documented in different regions (Kettle, 1995). Furthermore, incidental infestations in non-traditional hosts, such as dogs, have also been reported, highlighting the adaptive potential of the parasite under certain environmental conditions (McGarry et al., 2012).
       
Despite the growing body of research on O. ovis, significant gaps remain in understanding its epidemiological occurrence and associated host and clinical characteristics in specific endemic areas. In Saudi Arabia, previous investigations have addressed its occurrence in several regions (Alahmed, 2000; Hanan, 2013) and Metwally et al., (2021) investigated the prevalence and molecular characteristics of O. ovis in sheep and goats from Riyadh. However, information specifically from sheep examined at Al-Kharj slaughterhouses remains limited, particularly regarding associations of infestation with age, breed and season during the observed study period and its accompanying clinical signs. Furthermore, information on serum mineral concentrations in relation to O. ovis infestation is limited. Calcium, phosphorus and potassium are important for several physiological functions and their assessment may provide additional information on systemic alterations associated with infestation. Therefore, these minerals were evaluated to determine whether their serum concentrations differed between infested and non-infested sheep. This lack of localized epidemiological and biochemical information limits the development of targeted and evidence-based control strategies. Therefore, the present study aims to quantitatively assess the frequency of O. ovis infestation and its associations with age, breed and season among sheep examined at Al-Kharj slaughterhouses from December 2024 to August 2025. The study also aimed to describe the observed clinical signs and to compare serum calcium, phosphorus and potassium concentrations between infested and non-infested sheep, providing baseline data that may support improved surveillance and control programs in the region.

Study area
 
This study was conducted from December 2024 to August 2025 in Al-Kharj, located southeast of Riyadh and administratively affiliated with the Riyadh Region Fig 1. The area is characterized by a hot desert climate, with hot and dry summers, comparatively cooler winters and generally low and irregular rainfall, National Center for Meteorology (NCM, 2026). These climatic characteristics are provided as general background information for the study area and should not be interpreted as weather measurements recorded during the December 2024-August 2025 survey. Weather and climatic variables were not evaluated as explanatory factors for O. ovis infestation in the present study.


 
Animals
 
In the slaughterhouse
 
A survey of O. ovis infestation was conducted weekly from December 2024 to August 2025 at one slaughterhouse in Al-Kharj. A total of 480 sheep heads were examined for the presence of O. ovis larvae. The study included all eligible sheep available during the scheduled visits, without selection based on the presence or absence of clinical signs. No additional clinically suspect animals were recruited outside this sampling procedure. Data regarding breed, age and sex were recorded for each animal. Based on dentition, sheep aged two years or younger (≤2 years) were assigned to the younger age group, whereas those older than two years (>2 years) were assigned to the older age group. The individual sheep was considered the observational unit and animal identification numbers or identification marks were used to prevent duplicate inclusion. Infestation status was determined by the detection of O. ovis larvae during post-mortem examination. Information regarding previous antiparasitic treatment was unavailable.
 
Clinical assessment for suspected Oestrus ovis infestation: Ante-mortem inspection
 
Sheep presented for slaughter were subjected to ante-mortem examination for the detection of clinical signs consistent with suspected O. ovis larval infestation. Clinical observations were performed by a single examiner during routine ante-mortem inspection without a predefined fixed observation period. Clinical assessment focused on respiratory and nasal manifestations, including sneezing, serous nasal discharge, mucous nasal discharge, mucopurulent nasal discharge and blood-tinged nasal discharge. Animals exhibiting one or more of these clinical signs were classified as suspected cases and were clearly identified for subsequent post-mortem confirmation following slaughter. However, these clinical signs were considered suggestive rather than diagnostic of O. ovis infestation. All 480 sheep, regardless of their clinical presentation, underwent post-mortem examination for the recovery and morphological identification of O. ovis larvae. Animal identification numbers or identification marks were used to link ante-mortem observations, age, breed and post-mortem findings. Serum mineral results were also linked to the corresponding animals using the same identification system.
 
Examination procedures and larvae collection in the slaughterhouse: Post-mortem inspection
 
Post-mortem examination was performed on sheep heads at the slaughterhouse to detect and recover O. ovis larvae. The nasal cavities and frontal sinuses were carefully opened by longitudinally sectioning the skull along the dorsoventral plane using a sharp handsaw. Both nasal cavities and frontal sinuses were examined. The mucosal surfaces of the nasal septum, nasal passages and associated sinus regions were thoroughly inspected. Larval recovery was performed by direct visual inspection without additional rinsing or sieving procedures. Any O. ovis larvae attached to the mucosa were gently detached using forceps and collected for further analysis. Recovered larvae were immediately preserved in 70% ethanol for subsequent morphological examination. The preserved larvae were then transported to the Parasitology Laboratory, Department of Zoology, King Saud University, for further analysis. Identification and staging were carried out based on established morphological criteria using a stereomicroscope, with examination of external morphological characteristics, including body segmentation, cuticular spines and posterior spiracular plates. Larvae were classified into first, second and third instar stages (Smith, 1989). However, the available records documented the presence of second-and third-instar larvae (L2 and L3), without quantitative counts for each developmental stage. The presence of morphologically identified larvae in an examined sheep head was considered confirmation of infestation and each sheep was counted once when calculating infestation frequency, regardless of the number of larvae recovered.
 
Blood sample collection and analysis of biochemical parameters
 
Blood samples were aseptically collected from the jugular vein of 32 sheep (16 infested and 16 non-infested) using sterile 5 mL disposable syringes. The sheep were randomly selected and group classification was based on the presence or absence of Oestrus ovis larvae during examination. The collected blood was immediately transferred into plain tubes and transported under refrigerated conditions to the laboratory. Serum was separated by centrifugation at 3,000 rpm for 10 minutes and used for subsequent biochemical analysis. All samples were processed within four hours of collection to ensure optimal sample integrity and reliability of results. Serum concentrations of calcium and potassium were determined using a commercial veterinary diagnostic kit (Equine Panel, Skyla Corporation, Hsinchu, Taiwan), whereas inorganic phosphorus was measured using a separate skyla Ca/PHOS Single Assay Cartridge (PN 900-206, Revision A), according to the manufacturer’s instructions.
 
Statistical analysis
 
Statistical analysis was performed using IBM SPSS Statistics version 27.0. Categorical data were analyzed using pearson’s chi-square test or Fisher’s exact test, as appropriate. Age and breed associations were assessed using unadjusted analyses, while seasonal data were summarized descriptively. Cochran’s Q test was applied to paired binary clinical-sign data from 55 infested sheep. Mineral data were expressed as mean ± SD; normality and variance homogeneity were assessed using Shapiro–Wilk and Levene’s tests, respectively and groups were compared using the independent-samples t-test or Welch’s t-test when appropriate (16 infested and 16 non-infested sheep). Mineral comparisons were unadjusted. Statistical significance was set at P<0.05.

Due to the limited information available on the prevalence of ovine nasal myiasis caused by O. ovis in Al-Kharj, Saudi Arabia, a nine-month survey was conducted to assess the occurrence of infection among examined sheep. Of the 480 sheep examined, 55 were infected with O. ovis larvae, giving an overall prevalence of 11.46% (95% CI: 8.91-14.62%). Infection prevalence varied markedly between age groups, with sheep older than 2 years showing a significantly higher prevalence (39/160; 24.38%) than sheep aged 2 years or younger (16/320; 5.00%). This difference was highly statistically significant (Pearson’s χ² = 39.47, df = 1, P<0.001; unadjusted OR = 6.12, 95% CI: 3.30-11.37), indicating that infection was considerably more frequent among older sheep (Fig 2). However, this association was unadjusted and should not be interpreted as an independent effect of age. The overall prevalence recorded in the present study was higher than the 5.9% previously reported in Riyadh (Alahmed, 2000), but lower than the 22.3% reported by Metwally et al., (2021) in Riyadh and the 53.5% recorded in Jazan (Hanan, 2013). However, comparisons between studies should be interpreted cautiously because differences in host populations, sampling methods, study periods and diagnostic procedures may affect the reported prevalence. Previous parasitological investigations in Al-Kharj have also documented Echinococcus granulosus infection in stray dogs and local herbivores (Mares et al., 2023), highlighting the importance of continued parasitological surveillance in the region. The higher prevalence observed in sheep older than 2 years is consistent with previous reports showing greater O. ovis infection in older animals (Yilma and Genet, 2000; Gebremedhin, 2011; Hidalgo et al., 2015), whereas lower infection rates in younger sheep have also been reported (Silva et al., 2013).


       
The prevalence of ovine nasal myiasis differed between the two examined breeds. Harri sheep showed a higher infection prevalence (40/290; 13.79%) than Naimi sheep (15/190; 7.89%). The unadjusted Pearson’s chi-square test indicated a statistically significant difference between the two breeds (χ² = 3.94, P = 0.047; OR = 1.87, 95% CI: 1.00-3.48; Fig 3). However, this finding should be interpreted cautiously because the statistical significance was marginal. Breed-related variation in O. ovis infestation has also been reported in previous epidemiological studies. Mohammed and Soor (2025) observed differences in infestation prevalence among sheep of different breed origins and suggested that environmental adaptation, genetic background, previous exposure and stress may contribute to such variation. Similarly, Metwally et al., (2021) indicated that differences in O. ovis prevalence may be influenced by breed, geographical location and climatic conditions. However, because direct comparisons between harri and naimi sheep are limited, the higher prevalence observed in Harri sheep in the present study should be interpreted cautiously. Moreover, unequal age, seasonal and animal-source distributions between breeds may have confounded this association, while genetic and stress-related factors were not directly assessed in the present study.


       
The prevalence of ovine nasal myiasis varied significantly according to season. Infection was highest in spring (26.88%), followed by winter (5.63%) and summer (1.88%). The seasonal difference was highly significant (χ² = 57.33, P<0.001). Spring showed a significantly higher prevalence than both winter and summer, whereas no statistically significant difference was detected between winter and summer (Fig 4). This pronounced spring peak is consistent with Alahmed (2004), who reported the highest O. ovis infestation in March and greater larval abundance during spring in the Riyadh region. Similarly, Arslan et al., (2009) recorded the highest prevalence in spring in Turkey, while Papadopoulos et al., (2010) observed greater infection during early spring and autumn in Greece. In contrast, Metwally et al., (2021) found no significant seasonal effect on infestation prevalence in sheep from Riyadh, whereas Hanan (2013) reported a major peak during January in Jazan. These variations may reflect differences in local climatic and ecological conditions. The very low prevalence observed during summer in the present study may be related to the extremely hot and dry conditions of central Saudi Arabia, which may limit adult fly activity and parasite development. However, climatic conditions and fly activity were not directly measured and differences in flock origin, age, breed composition, recruitment, or previous treatment across seasons may also have influenced the observed pattern. Furthermore, autumn was not included in the sampling period and only one observation period was available for each sampled season.


       
Following the epidemiological assessment, the recovered larvae were examined morphologically to assess their developmental stages based on qualitative morphological characteristics. Recovered O. ovis larvae were identified based on stage-specific morphological features. Second-instar larvae (L2) were white, relatively smaller and showed distinct segmentation, cuticular spines and prominent mouth hooks (Fig 6 A-B). Third-instar larvae (L3) were larger, robust and yellow to light brown, with well-developed segmentation, strong cuticular spines and paired mouth hooks. These characteristics were recorded during morphological examination; Fig 6 (A-C) illustrates the gross appearance and coloration of the recovered larvae rather than their fine diagnostic structures. No morphometric measurements were performed. Gross examination of the nasal cavities and sinuses revealed larvae associated with visible mucosal inflammation and mucous exudate around their attachment sites (Fig 5 A-B-C). These findings were based on macroscopic observations without histopathological confirmation.




       
Among infected sheep (n = 55), mucous nasal discharge was the most commonly observed clinical sign (25/55; 45.45%), followed by sneezing (15/55; 27.27%), mucopurulent nasal discharge (8/55; 14.55%) and blood-tinged nasal discharge (3/55; 5.45%). Clinical signs could overlap and 12 sheep (21.82%) had no documented signs among these four categories. The distribution of the recorded clinical signs differed significantly (Cochran’s Q = 23.89, df = 3, P<0.001), with mucous nasal discharge representing the predominant clinical finding (Fig 7). Holm-adjusted pairwise comparisons showed significant differences between mucous and mucopurulent discharge (P = 0.006), mucous and blood-tinged discharge (P<0.001) and sneezing and blood-tinged discharge (P = 0.002), whereas the remaining comparisons were not significant. These observations are consistent with previous studies identifying nasal discharge and sneezing as common clinical manifestations of O. ovis infestation (Angulo‐Valadez et al., 2011; Schenkel et al., 2012). Similarly, Prabhu et al., (2019) reported sneezing, seromucous nasal discharge and epistaxis, whereas Bello et al., (2022) documented mucous and mucopurulent nasal discharges in naturally infected sheep. These signs may be associated with irritation and inflammation of the nasal mucosa caused by larval attachment and movement within the nasal passages.   

                    

Serum mineral concentrations differed between infected and non-infected sheep (n = 16 per group). Calcium levels were significantly lower in infected animals (8.26±1.00 mg/dL) than in non-infected animals (9.60±0.73 mg/dL; P<0.001). Similarly, serum potassium was markedly lower in infected sheep (3.04±0.29 mmol/L) compared with non-infected sheep (4.73±0.45 mmol/L; P<0.001). In contrast, phosphorus showed only a slight, statistically non-significant between-group difference (6.77 ±0.96 vs. 7.03 ± 1.14 mg/dL; P = 0.488; Fig 8-9-10). Although information on macro-mineral alterations during O. ovis infestation is limited, previous studies have demonstrated systemic physiological disturbances associated with oestrosis. Attia et al., (2020) reported oxidative stress and reduced serum zinc in O. ovis-infested sheep, while Ipek et al. (2023) also demonstrated increased oxidative stress in naturally infected animals. Similar mineral disturbances have been reported in other parasitic and ectoparasitic infestations, including reduced serum calcium in lice-infested sheep (Deger et al., 2002). Similarly, Mares et al., (2026) reported significantly reduced serum calcium concentrations in sheep naturally infected with Theileria spp. in Al-Kharj, Saudi Arabia, providing further evidence that mineral alterations may accompany different parasitic infections in sheep. Moreover, parasitic infections may reduce feed intake and alter mineral absorption and utilization (Bown et al., 1989; Coop and Holmes, 1996). Therefore, the reductions in calcium and potassium observed in the present study may reflect systemic stress or altered nutritional status; however, these mechanisms were not directly investigated. As the study was cross-sectional and the groups were not matched for potential confounding factors, the observed differences cannot be attributed solely to O. ovis infestation. Furthermore, the lower mean calcium concentration does not necessarily indicate clinical hypocalcemia.

The study detected O. ovis infestation in 11.46% (55/480) of sheep examined at an Al-Kharj slaughterhouse during the nine-month survey (December 2024-August 2025). As animal origins were not documented, the findings cannot establish local transmission or represent the wider sheep population. Further large-scale investigations are recommended to better assess the seasonal dynamics and economic impact of oestrosis in the region. The findings support further evaluation of locally appropriate surveillance and control strategies.

This work was funded by Ongoing Research Funding Program, (ORF-2026-2193), King Saud University, Riyadh, Saudi Arabia.

The authors declare that they have no conflicts of interest related to this study.