AAVS_13_s1

INTRODUCTION

Ovine brucellosis is primarily caused by two bacterial species: Brucella melitensis and B. ovis, despite small domestic ruminants being susceptible to other B. species. B. melitensis is predominantly recognised as a reproductive disorder in ewes, which is characterized by abortion and stillbirth in ewes and occasionally by epididymo-orchitis in rams (Rossetti et al., 2022). In contrast, another type of ovine brucellosis results from infection by B. ovis; it predominantly manifests as epididymitis in rams, occasionally resulting in abortions and neonatal mortality (Elderbrook et al., 2019).

Generally, antibody detection tests (serology) remain the most cost-effective method for the screening and detection of infected herds, especially under resource-limited settings (Legesse et al., 2023; Islam et al., 2025). In many epidemiological investigations, indirect ELISA is the method of choice because of its high specificity and sensitivity (Xu et al., 2020; Bulashev and Eskendirova, 2023; Legesse et al., 2023). Major variations exist in the occurrence of ovine brucellosis between countries and between parts of the world. According to a meta-analysis and systematic review, the overall seroprevalence of sheep in America, Africa, and Asia was 6.2%; the Americas had the lowest prevalence at 1.9%, while Africa had the highest at 8.5% (Wang et al., 2023). Several studies have established the issue of ovine brucellosis in various Iraqi provinces (Al-Farwachi et al., 2010; Al-Khafaji and Al-Farwachi, 2012; Al-Tae and Al-Samarrae, 2013; Al-Farwachi et al., 2019; Abdulhameed et al., 2020).

Although previous studies have assessed prevalence of brucellosis in Mosul city , this study is the first to simultaneously compare multiple serological tests (C-ELISA and I-ELISA) and evaluate the seroprevalence of antibodies against both B. ovis and other Brucella species in the same sheep flocks. Additionally, it identifies and compares the associated risk factors for each Brucella species, providing a comprehensive and novel understanding of ovine brucellosis in the region, despite the implementation of vaccination programs.

MATERIALS AND METHODS

Study area

The study was conducted in Mosul, a major city in northern Iraq situated 400 km north of Baghdad on the Tigris River. The metropolitan area covers approximately 32,308 square kilometers. Sheep are extensively raised in the Mosul region due to its temperate environment and the availability of natural pastures. Also, the city is a transit point for animals imported from countries neighboring Iraq.

This increases the risk of disease introduction into small ruminant population. Informal animal trade poses a significant risk for disease introduction. Additionally, the internal movement of sheep and goats between neighboring regions poses a risk of disease transmission. The study was carried out in several regions of the Mosul city , including Bazwaia, Kokjali, Bab Shamash, Veterinary Teaching Hospital, University of Mosul, between January 2024 to January 2025.

Twenty-five flocks of sheep were used in a cross-sectional study in Mosul, Iraq. The number of farms selected was proportional to the livestock population in the area of study, and the sample size within each farm and for different age groups within each farm was determined proportionally. Approximately 10% of animals from each flock were randomly sampled, with statistical analyses accounting for differences in flock size. To obtain an estimate with 5% precision and 95% confidence, and an estimated illness prevalence of 25 %. The sample size was calculated as:

n = Z²P(1-P)/d² (Charan and Biswas, 2021)

Z= Value of the normal distribution for a 95% confidence level; P= Expected prevalence; d= Absolute error; N= 289.

To improve the precision of prevalence estimates, accommodate potential sample attrition, and allow for accurate subgroup analysis across variables such as age, sex, and management techniques. A total of 400 serum samples were collected from non-vaccinated adult sheep (1.5-7 years old), comprising 320 ewes and 80 rams. The sample information, such as age, sex, origin and abortion history was documented.

Animal sampling

Five milliliters of venous blood was collected from each animal in a sterile, plain red-top tube and allowed to clot. The samples were transported on ice to the diagnostic laboratory of the College of Veterinary Medicine, University of Mosul. Serum was harvested after centrifugation (at 3000 rpm for 10 minutes) and stored at -20°C until tested.

Serological tests

All sera were tested by the following serological tests. The Rose Bengal test RBT (Institut Pourquier, Montpellier, France) was performed as described by Alton et al. (1988) and competitive ELISA (C-ELISA) kits (Svanovir®, Sweden). B. suis, B. melitensis, and B. abortus antibodies were detected by C-ELISA based on the smooth lipopolysaccharide (s-LPS) antigen and indirect ELISA (I-ELISA; IDEXX B. ovis Ab Test kit; Idexx Montpellier, France). After setting and incubating (Memmert®, Germany), the tests were washed (Biochek, USA). The optical density at 450 nm was was measured using an ELISA microplate reader. The S/P (sample-to-positive) ratio for each specimen was calculated according to the manufacturer’s instructions.

Statistical analysis

The data were analyzed by the Chi-square test and odds ratios (OR) with 95% confidence interval (95% CI) in SPSS V25 at a significance level of P≤ 0.05.

RESULTS AND DISCUSSION

In this investigation, 97 sera (24.3%) tested positive for the C-ELISA, which detects antibodies against Br. suis, Br. melitensis, and Br. abortus, and 62 sera (15.5%) tested positive for the I-ELISA, which detects antibodies against Br. ovis as shown in Table 1. However, 33/400 sera (8.3%) tested positive for both ELISA kits, as shown in Figure 1. Overall seroprevalence was 37.8% (151/400) with a 95% confidence interval of 31.8 to 44.3. In contrast, the Rose Bengal test identified antibodies in 83 sera (20.8%), with a 95% CI of 16.5 to 25.7 as shown in Figure 1.

Table 1: Prevalence of Brucella spp. antibodies among sheep in Mosul City, Iraq (N= 400).

Tests	Number of positive sera (%)	95% confidence interval (95%CI)	Chi-squared	P value
C-ELISA	97 (24.3)	19.7 to 29.6	9.704	P < 0.001
I-ELISA	62 (15.5)	11.9 to 19.9	9.704	P < 0.001

Ovine brucellosis is widely recognised as a considerable global challenge for sheep producers, particularly in areas that utilise extensive management practices for livestock rearing. Surveillance and vaccination, together with controlling animal movement, are the main measures to control brucellosis in livestock. The co-circulation of multiple B. species noticed in this study reflects Iraq’s complex brucellosis epidemiology. Risk factors such as unrestricted animal movement, ineffective immunisation programs, traditional husbandry practices, and a lack of awareness all contribute to the persistence of illness in livestock. Additionally, efforts to control the disease are further restricted by diagnostic difficulties such as serological cross-reactivity and inadequate disease reporting.

Compared to previously reported rates in Iraq, the seroprevalence of brucellosis in sheep in the current study was 37.8%, which is considered high. Lower prevalence has been observed in a number of previous investigations. Alshwany and Robertson (2018) reported a prevalence of 6.9% in Kirkuk province, but Ilyas et al. (2024) recorded a prevalence of 10.6% in Baghdad. According to various research, the prevalence in northern Iraq (Kurdistan Region), especially in Duhok, is moderately high, ranging from 31.7% to 35.4% (Al-Hamada et al., 2021; Ameen et al., 2023). Another study found that the Diyala Governorate had a higher prevalence rate of 59.5% (Al-Busultan et al., 2018).

The higher prevalence identified in this study could be attributable to factors such as poor vaccination coverage, inadequate biosecurity measures, and traditional husbandry practices. Iraq currently lacks a program to vaccinate cattle and buffaloes against brucellosis, despite the fact that the Rav1 vaccine is widely used to vaccinate sheep. Furthermore, the prevalence of stray dogs in sheep flocks plays a substantial role in disease transmission (Gharekhani et al., 2019; Mahmmodi et al., 2025).

This is supported by our findings, which identified contact with cattle and stray dogs as significant risk factors. Furthermore, unrestricted animal movement, including the importation of sheep from various countries, contributes to disease spread.

The current study demonstrated a significant prevalence of brucellosis among the animals under study in Iraq using various serological assays. In particular, 62 (15.5%) serum samples tested positive by I-ELISA, which targets B. ovis, while 97 (24.3%) and 83(20.8%) samples tested positive by C-ELISA and Rose Bengal test respectively, which detect antibodies against B. melitensis, B. abortus, and B. suis. This difference can be explained by the characteristics of the tests. C-ELISA is generally more sensitive and specific, detecting lower antibody levels and providing better differentiation between vaccinated and naturally infected animals. In contrast, RBT is a rapid screening test that may miss low-titer infections or early-stage seroconversion, leading to slightly lower prevalence estimates (Trangadia et al., 2015).

Higher seroprevalence detected by C-ELISA implies the presence of smooth Brucella species (Br. abortus, Br. melitensis, and Br. suis) in the examined population. This is particularly concerning in rural areas where close animal-human contact is common and consumption of raw dairy products is widespread. Previous Iraqi investigations have found human brucellosis to be prevalent, with Br. melitensis identified as the principal cause of human infections (Hou et al., 2013). The average yearly incidence of human brucellosis in Iraq was 41.88 cases per 100,000 individuals, with higher rates in Kurdistan (Hou et al., 2013). Research in Duhok discovered a 7% prevalence of brucellosis among children, highlighting the need for focused public health interventions (Alamian and Dadar, 2020).

In the present study, the seroprevalence of anti-Brucella ovis antibodies was lower than that seen for other Brucella species in this investigation. This finding may be explained by several factors. First, B. ovis has a restricted host specificity, with transmission occurring almost exclusively among rams and through venereal routes, whereas other Brucella species can spread more efficiently through horizontal and vertical pathways (Elderbrook et al., 2019).

Al-Farwachi et al. (2019) documented a Br. ovis seroprevalence of 4.8% among rams with epididymo-orchitis in Mosul city, northern Iraq. Similarly, in Karbala province, a study utilizing PCR on epididymal tissues from slaughtered rams found an overall B. ovis prevalence of 5%, with a significantly higher detection rate of 18% among animals (Wedajo et al., 2015). The higher seroprevalence of B. ovis observed in our study (15.5%) compared to the 4.8% reported by Al-Farwachi et al. (2019) may be attributed to several factors, including differences in sample size, animal selection criteria, variations in flock management and biosecurity measures.

Generally, younger animals, their mixing with other species, the presence of stray dogs among sheep flocks, abortion, and orchitis showed a statistically significant association with Br. spp. seropositivity compared to the other factors included in this study.

A statistical analysis of risk factors for Brucella spp. seropositivity in sheep demonstrated that ewes with a history of abortion were six times more likely to be seropositive for Brucella spp. than those without such a history (P < 0.001) as shown in Table 2. Ewes with a history of abortion had a prevalence of 43.0% ( P < 0.001), compared to 11.0% in those without abortion history. This is consistent with the known pathogenesis of Brucella spp., which are often implicated in reproductive disorders. The pathogenicity of Brucella is closely linked to its ability to localize and multiply in the reproductive organs, especially the uterus and placenta. A key factor contributing to this is the presence of erythritol, a sugar alcohol highly concentrated in the placenta of ruminants, which promotes the growth of Br. Species. Brucella induce placentitis, necrosis of placental tissue, and restriction of foetal development, ultimately leading to abortion (Narouei et al., 2022).

In the present study, rams with orchitis had a significantly higher rate of anti-Brucella ovis antibodies (28.9%) compared to those without lesions (7.1%), with an odds ratio of 5.3 (95% CI: 1.3–20.8, P = 0.01) as shown in Table 2. This finding indicates that testicular inflammation is an important clinical marker of Brucella ovis infection. The organism has a well-documented tropism for the male genital tract, where it causes epididymitis and orchitis, resulting in reduced semen quality and impaired fertility (Hou et al., 2013; Elderbrook et al., 2019; Rossetti et al., 2022). Further supporting the role of reproductive tract infections in disease dynamics. Both orchitis and abortion serve not only as disease indicators but may also promote pathogen transmission within flocks, especially in systems lacking proper reproductive management (Rossetti et al., 2022).

The current investigation showed a significant association between the presence of stray dogs and increased seropositivity in sheep flocks. Specifically, flocks exposed to stray dogs were 3.4 times more likely to be seropositive for Brucella spp. than those not exposed (P < 0.001), as

Table 2: Animal factors-wise and the seroprevalence of Brucella spp. and B. ovis antibodies among sheep at Mosul city governorate using ELISA kits.

Variables	Criteria	Number of samples	Brucella spp.	Odds ratio (95% CI)	P value	Brucella ovis	Odds ratio (95% CI)	P value
Variables	Criteria	Number of samples	Seropostive (%)	Odds ratio (95% CI)	P value	Seropostive(%)	Odds ratio (95% CI)	P value
Age(years)	1.5-3	120	38(31.7)	2.4(1.4 to 4.2)	0.001	26(21.7)	2.5( 1.3 to 4.9)	0.004
	3- 5	120	33(27.5)	1.95(1.0 to 3.5)	0.01	20(16.7)	1.8( 1.0 to 3.6)	0.05
	> 5	160	26(16.3)	Reference		16(10.0)	Reference
Sex	Ewes	320	72(22.5)	0.7(0.429 to 1.2)	0. 1	48(15.0)	Reference
Sex	Rams	80	25(31.3)	Reference		14(17.5)	0.7(0.3 to1.3)	0.11
Managements	Sheep and cattle	120	41(34.2)	2.3 ( 1.4 to 3.9)	0.001	5(4.1)	Reference
	Sheep and goats	100	23(23.0)	1.3( 0.7 , 2.4)	0.2	18(18.0)	5.0(1.8 to14.1)	0.001
	Sheep only	180	33(18.3)	Reference		39(21.7)	3.4(2.4 to 16.7)	0.00008
Stray dogs	Yes	166	62(37.3)	3.4( 2.1 to 5.5)	0.0	30(18.0)	1.5( 0.9 to 2.5 )	0.07
Stray dogs	No	234	35(15.0)	Reference		32(13.7)	Reference
Abortion	Yes	130	56(43.0)	6.0(3.4 to 10.8)	0.0	22(16.9)	0.8(0.4,1.4)	0.2
Abortion	No	190	21(11.0)	Reference		26(13.7)	Reference
Orchitis	Yes	38	11(28.9)	0.81 (0.31to 2.1)	0.67	11(28.9)	5.3 (1.3to 20.8)	0.01
Orchitis	No	42	14(33.3)	Reference		3(7.1)	Reference

(95% CI= Confidence Interval).

shown in Table 2, indicating a very significant association. Infected dogs can transmit Br. abortus and Br. melitensis to specific hosts, humans, and other animal species.Furthermore, infected dogs can play a vital role in the persistence of brucellosis in ruminants (Gharekhani et al., 2019; Mahmmodi et al., 2025).

In farms, the role of stray dogs in the epidemiology of brucellosis can be twofold. They may act as mechanical carriers by contacting aborted materials, placenta, or contaminated environments. Without being infected themselves, they can subsequently spread Brucella organisms to healthy flocks. This increases infection rates and compromises biosecurity measures. On the other hand, dogs can also become biologically infected by Brucella canis and occasionally B. melitensis, indicating their potential role as true reservoirs .Effective control strategies must include systematic screening of dogs associated with herds and implementing vaccination protocols for both sheep and dogs is essential for managing brucellosis outbreaks (Hou et al., 2013; Alamian and Dadar, 2020).

The highest seroprevalence for Brucella spp. was observed in the 1.5–3 years age group (31.7%), with an odds ratio (OR) of 2.4 (95% CI: 1.4–4.2, P< 0.001). The 3–5 years old group also showed increased risk (27.5%; OR = 1.95, 95% CI: 1.094–3.49, (P < 0.01), compared to animals older than 5 years (16.3%), which were used as the reference group, as showed in Table 2.

Young, sexually mature animals are more susceptible, possibly due to increased exposure or immunological naïveté. while older animals appear more resistant, probably due to previous exposure conferring some immunity. Similar findings have been reported in previous epidemiological studies of ovine brucellosis (Wedajo et al., 2015; Narouei et al., 2022).

Mohammed et al. (2017) and Sorsa et al. (2022) reported higher seroprevalence in older age groups than in younger animals, even though the difference was not statistically significant. In contrast, Hussen et al. (2023) reported a higher seroprevalence in the old age group than in the medium and young age groups.

Among sheep aged 1–3 years, 26 animals (21.7%) tested seropositive for Brucella ovis with an odds ratio of 2.5(95% CI: 1.3–4.9; P < 0.004), indicating a significantly higher likelihood of seropositivity in this age group as showed in Table 2. Indicates that that Brucella ovis seroprevalence correlates with sexual activity. This increased seropositivity in young adults may be attributed to several factors. First, sexually mature animals are more likely to be exposed to infection during mating . Second, animals in this age group may have relatively naïve immune systems, lacking previous exposure that could confer partial immunity. These results align with other studies reporting higher seroprevalence in younger, sexually active sheep compared to older animals, suggesting that age and sexual maturity are key determinants in the epidemiology of ovine brucellosis (Al-Farwachi et al., 2019; Elderbrook et al., 2019).

Herd composition was another significant factor influencing the epidemiology of ovine brucellosis. Sheep raised in mixed flocks with cattle showed a significantly higher rate of anti-Brucella spp. antibodies(34.2%) compared to those in sheep-only flocks as showed in Table 2.

Also, Br. ovis seropositivity was found to be significantly influenced by management techniques. Sheep raised with goats had a significantly higher prevalence (18.0%) than those raised with cattle (4.1%) (OR = 5.0, 95% CI: 1.8-14.1, P < 0.001). Similarly, sheep raised in sheep-only systems showed a high seropositivity rate (21.7%) and were at higher risk (OR = 3.4, 95% CI: 2.428-16.66, P < 0.00008) as in Table 2. While young animals aged 1.5–3 years demonstrated the lowest odds at 2.5 as shown in Table 2.

These findings were consistent with previous studies demonstrating that multi-species farming systems facilitate the cross-species transmission of Brucella spp., particularly under extensive management or shared grazing conditions where biosecurity is often inadequate (Godfroid et al., 2013; Sibhat et al., 2024).

Mixed-species rearing is still frequent across Iraq due to traditional herding methods, which increases the possibility of interspecies transmission. Furthermore, shared water sources, the same grazing fields, and inadequate species separation during lambing and kidding seasons all increase the risk of transmission. As a result, effective control strategies in endemic areas should include species-specific interventions while also addressing the complex dynamics brought on by mixed-species livestock management (Martindah et al., 2025). In contrast, sheep-only herds presented a lower, yet still notable, odds ratio of 23%, indicating that while mixed herds increase risk, brucellosis remains prevalent even in single-species flocks, likely due to environmental contamination or animal movement.

CONCLUSIONS AND RECOMMENDATIONS

Brucellosis is still a prevalent disease in sheep flocks, highlighting the ongoing risk of transmission in mixed-species livestock systems. Variable detection rates were observed, with minimal overlap between the results obtained from the C-ELISA and I-ELISA kits. Ewe abortion history, the presence of stray dogs, co-management with goats or cattle, younger age, and ram orchitis are key risk factors, highlighting areas that require targeted preventive measures.

ACKNOWLEDGMENT

This study is approved by the College of Veterinary Medicine, University of Mosul, Iraq.

NOVELTY STATEMENT

This is the first study that provides updated epidemiological data on brucellosis in sheep flocks in Iraq using a mix of serological assays (RBT, C-ELISA, and I-ELISA). The research reveals the minimal overlap between different ELISA kits, demonstrating major disparities in diagnostic performance that have not been previously observed in the region. Furthermore, the identification of particular risk factors, such as ewe abortion history, ram orchitis, co-management with cattle or goats, and the presence of stray dogs, provides new insights into the transmission dynamics of Brucella spp. and B. ovis in mixed-species livestock systems. This study provides crucial data and provides significant guidance for focused control and preventative programs.

AUTHOR’S CONTRIBUTION

All authors contributed equally.

Ethical consideration

Ethical issues (including plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy) have been checked by all the authors. All required institutional ethical approvals were obtained before this study was conducted (Approval Date18/10/2024 and Number UM.VET.2024.101).

Generative AI and AI-assisted technology statement

All authors of this work declare that generative AI technologies including large language models (e.g., ChatGPT, Copilot) and text-to-image generators were not utilized in any capacity during the preparation, writing, or editing of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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