Seroprevalence and risk factors: A comprehensive seroepidemiological study of epizootic hemorrhagic disease and bluetongue in Northwestern Tunisia

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The epidemiological situation of EHD in Tunisia remains poorly documented, despite several studies having been conducted on BT. To assess the seroprevalence of the EHD and BT in northern Tunisia, 394 bovine serum samples were collected and tested for anti-VP2 antibodies using a competitive ELISA. The seroprevalence of EHD and BT at the individual level was estimated at 51.2% and 81% respectively. Herd-level seroprevalence reached 93% for EHDV and 100% for BTV. No statistically significant differences in prevalence were observed between governorates for EHD (p-value = 0.169). However, the prevalence of BT across governorates was found to be statistically significant (p-value = 0.00000). A multivariable mixed-effects logistic regression were conducted to identify risk factors for EHD and BT. The final model revealed that risk factors associated with EHD and BT seroprevalence may include only age where an age-related increase in seroprevalence was noted for both diseases. Local animal husbandry practices and herd management were not found to be associated with the dynamics of the two diseases. The findings of this study highlight the geographical extent of the disease in the northwestern region and its associated risk factors. It is therefore imperative that further investigations be conducted on vectors and their abudance in order to gain a comprehensive understanding of the current situation. Epizootic hemorrhagic disease Bluetongue seroprevalence survey risk factors cattle Tunisia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The genus Orbivirus comprises several significant viruses that exert a detrimental effect on animal health. Vector-borne diseases caused by viruses such as epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) primarily affect livestock and wildlife, leading to severe outbreaks and significant economic losses in agriculture. Both viruses belong to the family Reoviridae and share morphological and structural similarities (Maclachlan & Osburn, 2004). As with other Orbivirus members, BTV and EHDV possess seven structural proteins (VP1 to VP7), forming two capsids. The VP2 protein, exposed on the surface of the virion, serves as the serotype-specific antigen (Wilson & Mellor, 2009).These diseases are recognized as notifiable by the World Organization for Animal Health (Jiménez-Cabello et al., 2023). The EHDV was detected for the first time in 1955 in North America (Mejri et al., 2018). According to literature, seven serotypes of EHDV have been identified, numbered 1–2 and 4–8 (Ben Hassine et al., 2024). BT was first recognized as a disease of cattle in 1933 (Joardar S.N, 2022). As of now, there have been 36 distinct serotypes of BTV identified. This includes 24 classical serotypes and 12 that are classified as atypical (Ries et al., 2020; Ries et al., 2021). Research has demonstrated how climate change exacerbates the distribution of such diseases, thereby enabling their emergence in regions where they were previously absent (Savini et al., 2011; Purse et al., 2005; Clavijo et al., 2002). For instance, the spread of BTV has been impacted by changing weather patterns affecting insect dynamics, thereby increasing the risk of outbreaks in susceptible animal populations (Brand et al., 2017). BTV and EHDV are transmitted by Culicoides biting midges, and their co-circulation is considered a common phenomenon (Savini et al., 2011). Tunisia is endemic to both viruses. The first incursion of BTV occurred in 1999, since then, multiple serotypes, including BTV-1, BTV-2, BTV-3, BTV-4, BTV-Y and BTV-26, have been identified (Kalthoum et al., 2022). The first outbreak of EHDV was documented in 2006, resulting in significant economic losses for various cattle farms due to high mortality and morbidity. The Tunisian EHDV strains were classified as serotype 6 (Ben Dhaou et al., 2016). In 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia (Sghaier et al., 2023). Despite neighbouring countries like Algeria showing a link between EHDV and BTV prevalence with vector distribution ( Culicoides imicola ) (Hafsa Madani et al., 2011), data on EHDV prevalence and risk factors in Tunisia remain scarce, although risk factors for BTV have been studied in the country and an overall seroprevalence of 40.1% was reported (Kalthoum et al., 2022). The present study aims to assess the prevalence and distribution of EHDV antibodies in organized farms in northwest Tunisia. By identifying risk factors, it intends to provide valuable insights into EHDV and BT transmission dynamics, contributing to effective disease management strategies. Materials and Methods Study area The study was conducted from 25 April to 19 May 2023, in four governorates in the north-western Tunisia : Jendouba, Beja, Siliana, and Kef (Fig. 1 ). These governorates were further subdivided into 76 districts and 744 sectors, covering an area of 16,267 km2, i.e. 10% of the total area of Tunisia with a total population of 1,210,000 inhabitants (Fig. 1 ). The study area is the rainiest region of the country, receiving more than 1,000 millimetres of rainfall per year. It is characterised by the presence of important watercourses and reservoirs. Sapmling design and serum collection The study population consisted of organized farms in the north-western region of Tunisia, comprising 31 cattle farms from both the private and state sectors, with a total population of 8,634 cattle. As this is the first serological survey targeting EHDV and BT in the region, all farms within the area were included in the study. The exhaustive list of animals across the 31 farms was provided by the regional veterinary services and the Office of Livestock and Pastures (OEP). No animals were euthanized or sacrificed during this study; only non-invasive blood sampling was conducted. The following parameters were used to calculate of the required sample size: An expected prevalence of 25%, based on histroical outbreaks data. A desired absolute precision of 15% A 5% risk of error (reference) . A total of 484 cattle were initially required. A proportional sampling approach was used to determine the number of cattle to be sampled per farm, ensuring that the sample accurately reflected the distribution of animals across the farms. To guarantee representativeness of our sample, the cattle to be sampled were randomly selected using the sampling frame. Only animals over six months of age were included in the study to avoid potential interference with passive immunity. It is important to note that the study exclusively enrolled cattle that had not previously been vaccinated against BTV. As no EHD vaccines are commercially available worldwide, this study provided an opportunity to assess the natural prevalence of EHD in the absence of vaccination. During the study, two farms initially scheduled to participate declined, reducing the final sample size to 396 cattle distributed across 29 farms. A 10 ml serum was collected from each animal, strictly adherence to animal welfare standards. Each sample was distinctly labeled using a predefined code accurate identification. All the samples were centrifuged to separate the serum and then, stored at -20°C. The stored samples were subsequently transported under controlled conditions to the Institute of Veterinary Research of Tunisia (IRVT) for serological analysis. Data collection The study was carried out between April and May 2023. Farms and animals data were collected using a structured questionnaire specifically developed for this study consisting of three main parts. The first part focused on farm-related information, including the farm name, geographical location, type of building and housing conditions, total number of cattle, and the history of BT-like diseases on the farm. The second part is dedicated to individual animals and collected details such as age, sex, and breed. The final section covered potential risk factors associated with exposure to the BTV and EHDV, such as the introduction of animals, the presence of stagnant water, and proximity to wetlands. An English version of the questionnaire is provided as Supplementary File S1. Data was collected using an application of the Kobotoolbox platform( www.kobotoolbox.org ). For the environmental risk factors, data on temperature were extracted from https://www.ogimet.com for the period ranged between 30 march 2020 and 31 may 2023. Detection of antibodies against BTV and EHDV All sera were tested using the competitive enzyme-linked immunosorbent assay (ELISA) for EHDV (ID Screen® EHDV Competition, IDVet, France) and the BTV ELISA to detect antibodies directed against the VP7 protein (c-ELISA) kit (ID SCREEN® Bluetongue Competition ELISA, IDVet, Grabels, France). Analyses were performed according to the manufacturer's instructions. Samples with an IP greater than 60% were considered positive, indicating the presence of specific antibodies against EHDV or BTV. Statistical analysis The extracted data was organized into an Excel spreadsheet. The age variable was transformed into categorical variable with three defined age classes: Class 1(6–12 months), Class 2 (13–24 months) and Class 3 (more than 24 months). Herd size was categorized into three classes using the quartile classification method (Ross, 2014). The seroprevalence of BTV and EHDV was estimated at both the herd and individual levels and the 95% confidence interval (CI) was calculated using the binomial exact method. The apparent herd prevalence was calculated as the proportion of positive herds among the total number of herds tested. The prevalence at the individual level was determined by dividing the number of positive animals by the total number of animals tested in that herd. Raster for temperature was created using the interpolation method and values on farm location were extracted using Arcgis function “extract values to point”. Raster for NDVI was extracted from https://www.copernicus.eu/en for the period between 30 april 2019 and 31 may 2023. Values on farm location were also extracted using Arcgis function “extract values to point”. Univarite and multivariable mixed-effects logistic regression were conducted to identify risk factors for EHD and BTV at herd and animal level. The status of EHD and BT infection was designated as the outcome variable. The explanatory variables considered for univariate and multivariate analysis, are as follows : history of BT-like diseases on the farm, type of building, distance from wetlands, presence of mosquitoes at the farm, presence of stagnant water on the farm, animal exchange with other farms, presence of wetlands close to the farm, age, sex, herd size, and breed. Associations between seroprevalence of EHD and BT and potential risk factors were initially assessed through univariable analysis and only variables with p < 0.2 were subsequently included in the logistic multiple-regression model. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated. The presence of multicollinearity among the variables was evaluated using generalized variance inflation factors (GVIFs). The Hosmer-Lemeshow goodness-of-fit test was performed on the final model. Furthermore, receiver operating characteristic (ROC) curves and area under the curve (AUC) values were calculated in order to assess the model’s predictive performance. All statistical analyses were performed using R software, and the maps were generated by ArcGIS version 10.4. Results Cattle and farm characteristics A total of 394 cattle, consisting of 386 females (97.9%) and 8 males (2.03%) belonging to 29 farms in four north-western governorates, were recruited for this study. As for the demographic characteristics of the sample, the age of the cattle ranged from 7 to 123 months with a mean of 40 months. The breed of the sample was predominantly Holstein constituting 85.02% (335/394) of the total. Other breeds including Montbeliarde, Swiss and Tarentaise accounted for 14.84% (59/394). For the gender, the sample is mainly of females accounting for 97.9% (386 out of 394). In term of age, the animals are categorised into three distinct age classes: 6–12 months, 13–24 months and over 25 months. The age group most represented is that of cattle over 25 months of age, representing 67.5% (266/394) of the sampled animals. The characteristics of the surveyd farms and cattle are presented in the Table 1 . Table 1 Characteristic of the surveyed famrs and sampled animals Variable Category Frequency Herd size Large [> 300] 5 (17.2%) Small [< 300] 24 (82.7%) Age class [ 6 – 11 ] 31 (7.8%) [ 12 – 24 ] 97 (24.6%) [25–123] 266 (67.5%) Sex Female 386 (97.9%) Male 8 (2,03%) Breed Holstein 335 (80.02%) Montbeliarde 12 (3.04%) Swiss 37 (9.3%) Tarentaise 10 (2.5%) Housing type Indoor 10 (2.5%) Semi-enclosed 384 (97.4%) Seroprevalence of the EHD and BT At the herd level, all the farms were tested positive for the BT (29/29 (100%)) while 93.1% (95% CI: 77.23–99.15%) (27/29) were tested positive for anti-EHDV antibodies. At the individual animal level, 198 out of 394 samples tested positive for EHD giving an overall seroprevalence rate of 50.3% (95% CI: 45.2%-55.3%). For BT, 81,2% (320/394) (95% CI: 77%-85%) of the tested animals were positive (Table 2 , Fig. 2 ). Table 2 Prevalence of EHDV and BT at the herd and animal level Disease Herd prevalence (%) Animal prevalence (%) EHDV 93.1% [77.2–99.2%] 50.3% [45.2–55.3%] BT 100% [88.1–100%] 81.2% [77–85%] The presence of dual exposure to BT and EHD was detected in 27 (93.1%) of the tested herds. At the individual level, dual exposure to BT and EHD was detected in 182 (46.2%) of the total number of cattle (Fig. 3 ). According to the governorate, 7 cattle out of 10 selected from the Kef governorate, were found to be positive to EHD representing the highest level of the seropositivity followed by Jendouba where 54.8% (91/166) of the cattle tested were positive. The lowest seroprevalence of EHD was recorded in Beja with 44.2% (61/138) of the cattle being seropositive. However, statistical analysis revealed no statistically significant differences between the governorates (p-value = 0.169). However, for BT, the results indicate that Siliana has the highest prevalence of BT with 92.5% (74/80), followed by Jendouba (82.5% (137/166), Beja (76.1% (105/138) and Kef (40% (4/10)) (Fig. 4 ). The observed variation in the prevalence of BT across governorates was found to be statistically significant (p-value = 0.00000). Risk factors of EHD and BT Univariate analysis revealed that the age and temperature were identified as significant predictors of BT. As demonstrated in Table 3 , the risk factors associated with a positive test result for EHD were distance from wetlands, age and the presence of wetlands (Table 3 ). The final model demonstrated that the age was the only significant predictor for both EHD and BT. Animals aged 25–123 months (Category 3) have significantly higher odds of seropositivity of BT compared to younger (OR = 5.30, 95% CI: 1.77–15.87, p = 0.002). While temperature showed a non-significant association with BT seropositivity (OR = 1.57, 95% CI: 0.91–2.73, p = 0.10). Regarding EHD, both category of age (Category 2 and 3) demonstrated significantly increased odds compared to younger animals (Category 2 :12–24 months; OR = 7.81, 95% CI: 1.54–39.52, p = 0.01, Category 3 :25–123 months; OR = 21.1, 95% CI: 4.38-102.11, p = 0.0000). Farms located less then 1 km from wetlands tended to have lower odds of EHD seropositivity, although, this association did not reach statistical significance (OR = 0.25, 95% CI: 0.059–1.11, p = 0.07) (Table 4 , Fig. 5 ). All other variables, including housing type, breed, mosquito presence in the farm, NDVI, stagnant water, and flock size, showed no significant associations with either disease in the univariate analysis. Table 3 Univariate analysis for the association between potential risk factors and BT and EHD seropositivity among cattle in Northwestern Tunisia Variables Category BT EHD OR [IC95%] p-value OR [IC95%] p-value Housing type Semi-enclosed 1.44 [0.06–34.42] 0.82 1.62 [0.09–28.14] 0.74 Breed Holstein 0.63 [0.15–2.70] 0.54 0.95 [0.29–3.09] 0.93 Distacne from wetlands Distance < 1 km 0.59 [0.17–2.05] 0.41 0.26 [0.06–1.02] 0.05 Presence of mosquitos in the farm Yes 0.51 [0.07–3.74] 0.51 0.81 [0.15–4.36] 0.81 NDVI NDVI (per unit) 1.07 [0.05–22.49] 0.97 0.85 [0.06–11.08] 0.9 Presence of stagnant water Yes 1.46 [0.15–14.53] 0.75 1.03 [0.13–8.20] 0.98 Age class Category 2 : [12–24 moths] 1.02 [0.33–3.14] 0.98 7.02 [1.98–24.87] 0.002 Category 3 : [25–123 months] 5.31 [1.77–15.87] 0.003 20.20 [5.91–69.03] 0.000 Flock size [> 300] 0.72 [0.20–2.54] 0.61 0.92 [0.30–2.78] 0.88 Temperature Temperature (per unit) 1.52 [0.94–2.47] 0.09 1.25 [0.79–1.97] 0.35 Table 4 Multivariate analysis for the association between potential risk factors and BT and EHD seropositivity among cattle in Northwestern Tunisia Variables Category BT EHD OR [IC95%] p-value OR [IC95%] p-value Distacne from wetlands Distance < 1 km - - 0.25 [0.059–1.11] 0.07 Age class Category 2 : [12–24 moths] - - 7.81 [1.54–39.52] 0.01 Category 3 : [25–123 months] 5.30 [1.77–15.87] 0.002 21.16 [4.38-102.11] 0.0001 Temperature Temperature (per unit) 1.57 [0.91–2.73] 0.10 - - Discussion In Tunisia, the surveillance system of vector-borne diseases such as BT and EHD relies only on the passive surveillance. Since the first report of EHD in Tunisia and according to the previous study, the northwestern region has been identified as a highly infected area. Both diseases (BT and EHD) can be transmitted by the same vector and cases of co-infection have been previously documented (Ben Hassine et al., 2024). To our knowledge, few studies have been considered the seroprevalence of the vector-borne diseases specially in the northwestern Tunisia. Therefore, the present study aims to assess the serporevalence of BT and EHD in organized farms in the northwestern Tunisia and to identify potential risk factors. The results of this study indicate the widespread of the BT and EHD in the northern Tunisia. The overall seroprevalence of EHD estimated animal-level in this study (50.3%) is considerably higher than that reported in previous studies. Mejri et al. (2018), reported a seroprevalence of 8.6% during a sentinel study conducted in cattle between 2013 and 2014, Ben Dhaou (2017) detected a seroprevalence of 5.3% in the governorate of Bizerte in 2012. This notable increase in seroprevalence may reflect a recent intensification in virus circulation and can be attributed to several factors such as favorable climatic conditions for vector activity and changes in vector competences, abundance and distribution (González-Recio at al.2025). It was demonstrated that significant changes have been observed in the pattern of disease and distribution of EHDV and an increase in the severity of the disease among bovine populations (Noronha et al., 2021; Jiménez-Cabello et al., 2023). Our results align with the findings of various authors across different countries : 57.87% in China, 57.1% USA (Northern Florida), 66.9% in Pakistan and 64% in Kenya (Gordon et al., 2017; Orange et al., 2021; Lv et al., 2023; Ishaq et al., 2025). In contrast, studies conducted in neighbouring and non-neighbouring countries reported a low seroprevalences of 1% and 9% in cattle in Libya in 2021, Algeria in 2011 and India in 2010, respectively (Boyer et al., 2010; Madani et al., 2011; Mahmoud et al., 2021 ). For BT, the overall seroprevalence at the individual level was very high (81.2%) in our study indicating a high degree of exposure of cattle in the northern Tunisia to the virus. This result suggests recent circulation of the virus of BT in the study area. The observed high seroprevalence in these farms is likely indicative of a substantial population of Culicoides vectors that is facilitated by favorable climatic conditions. As demonstrated in the relevant literature, comparable high seroprevalences of BT have been reported in Kenya (91.5%), Estern Sudan (92.9%) and Brazil (83.3%) (Chiuya et al., 2024; Abraheem et al., 2024; Neves et al., 2022). However, the seroprevalences of BT reported in southern Italy (43.6%), Iran (27.63%), Algeria (13.7%) and Peru (19.3%) were lower than our finding (Kardjadj et al., 2016; Ferrara et al., 2024, Bakhshesh et al., 2020; Navarro-Mamani et al., 2025). The present study indicated that 46.2% of the tested cattle were co-infected with both BT and EHD. This is considered a high prevalence in comparison to the studies conducted in Mayotte and USA, where 9.4% and 1.49% of the samples were positive for both diseases, respectively (Schroeder et al., 2013; Sailleau et al., 2012). However, it aligns with the findings of Min-Na Lv et al. in China in 2012–2013 (40.3%) (Lv et al., 2023). The presence of BT and EHD coinfections observed in our study can be attributed to several factors. The viruses that cause theses diseases are both transmitted by the same genus of hematophagous biting midges (Culicoides spp.) and when the ecological niches of the two diseases overlap (Lv et al., 2023). Consistent with other research (Boyer et al., 2010; Rivera et al., 2021), our results confirm that significant exposure to EHDV and BTV is associated with the abundance of Culicoides spp. in the region. The study revealed no significant differences in the EHD seroprevalence across governorates suggesting uniform exposure of cattle to this disease in the study area, wich may be due to similar environmental conditions and vector abundance. However, a significant variation in the seroprevalence of BT was highlighted, wich can be attributed to the differences in livestock species composition, breed susceptibility, and host and vector density (Chanda et al., 2019; Saminathan et al., 2020). Additionnaly, the seroprevalence of BT may be influenced by herd management practices, including vector control measures and grazing patterns (Beyen el al., 2025). C Based on the univariate and multivariate analysis, we studied the association between seroprevalence of BT and EHD and the following risks factors ; Housing type, breed, distacne from wetlands, presence of mosquitos in the farm, NDVI, presence of stagnant water, age class, flock size and temperature. The results revealed that only the age is a risk factor for both diseases in the final model, but with stronger effects observed for EHD, reflecting distinct epidemiological dynamics between the two infections. The age-related increase in seroprevalence observed for both viruses aligns with findings from previous studies, which attribute higher exposure over time to the cumulative effect of vector contact as cattle age (Ibrahim et al., 2014; Bachir Medrouh et al., 2024; Chiuya et al., 2024; Şevik M, 2022). This highlights the endemic nature of these pathogens in environments with a high prevalence of vectors. The absence of significant breed-specific differences in seroprevalence of the two vector-borne diseases, previous studies conducted overwide suggest that susceptibility to EHD and BT in endemic areas is primarily determined by vector exposure rather than genetic factors (Manavian et al., 2017; Silvia et al., 2023). Furthermore, the seroprevalence of both diseases was found not to be associated with housing type. This finding is not unexpected, given that Culicoides are indoor and outdoor vectors and seasonal variation in climatic conditions influences their indoor and outdoor activity patterns (Magliano et al., 2018; Groschupp et al., 2024). The other investiguated risk factors were found to be significantly associated with either disease. Despite the valuable data provided by this study, several limitations should be acknowledged. First, the study relied on cross-sectional serological data, which limits the ability to ascertain whether the viruses under investigation are recent or old circulation. Furthermore, environmental data used in this study such as temperature and NDVI were extracted from external sources and did not cover all the time periods of exposure, which may influence the association of these variables with BT and EHD seroprevalecne. Finally, the study did not include molecular detection of active infections or virus typing, which could provide deeper insights into circulating strains and the dynamics of co-infections. The findings of the present study enhance our comprehension of the epidemiological profiles of two vector-borne diseases (BT and EHD) in northern Tunisia, as well as the risk factors associated with their seroprevalences. The study provides valuable information for the control programmes of these diseases and for the adaptation of strategies in order to reduce the risk of their occurrence. Conclusion The present study highlights the widespread of BT and EHD in organised cattle farms across the northwesterern Tunisia. The high seroprevalence of both diseases indicates favourable environment for the vectors implicated in their transmission and spread. Although the study provided valuable data on the seroprevalence of the two diseases, it remains unclear whether these infections were occured. Future longitudinal studies incorporating molecular diagnostics and entomological surveillance are needed to better understand the transmission dynamics and temporal trends of BT and EHD in Tunisia. Declarations Acknowledgements The authors would like to thank the veterinarians and the staff of the organized farms, whose support made this work possible. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Authors contributions Imed Ben Slimen, Sana Kalthoum, Kaouther Guesmi, Mohamed Naceur Baccar : Conceptualization and methodology , formal analysis, writing-review and editing Imed Ben Slimen : writing-original draft preparation Aida Tlatli, Soufien Sghaier, Aida Megdich, Sonia Ben Hsan, Salma Hadouchi : Laboratory analysis Hanen Ncibi, Chafik Ben Salah, Ilyes Arfaoui, Mohamed Yahya Dalhoumi, Marwa Sallami, Raja Gharbi: investigation and data collection Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval ans consent to participate This study was reviewed and approved by the AnimalEthicsCommittee-NationalSchool of Veterinary Medicine,SidiThabet under the approval number CEEA-ENMV92/25 . Informed consent was obtained verbally from all participating animal owners after explaining the objectives of the study, and participation was entirely voluntary. No animals were euthanized or sacrificed during this study; only non-invasive blood sampling was performed. Consent to Publish declaration: Not applicable Competing interests The authors declare that they have no competing interests References Abraheem HH, Hussien MO, Elhassan AM, Enan KA, Musa AB, Ahmed SK, El Hussein ARM. Seroepidemiological Survey on Bluetongue Virus (BTV) among Cattle, Sheep, and Goats in Gadarif State, Eastern Sudan. Int J Microbiol. 2024;2024:7712412. 10.1155/2024/7712412. PMID: 39328248; PMCID: PMC11424855. Bakhshesh M, Otarod V, Fallah Mehrabadi MH. Large-scale seroprevalence and risk factors associated with Bluetongue virus in Iran. Prev Vet Med. 2020;179:104994. 10.1016/j.prevetmed.2020.104994. Epub 2020 Apr 30. PMID: 32402914. Ben Dhaou S. Etude de la maladie épizootique hémorragique en Tunisie. Sciences agricoles. Université Paris-Est; Faculté des sciences de Bizerte (Tunisie), 2017. Français. ffNNT: 2017PESC0023ff. fftel-01763049f. Ben Dhaou S, Sailleau C, Babay B, Viarouge C, Sghaier S, Zientara S, Hammami S, Bréard E. Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006. Acta Vet Hung . 2016;64(2):250 – 62. 10.1556/004.2016.025. PMID: 27342096. Ben Hassine T, García-Carrasco J-M, Sghaier S, Thabet S, Lorusso A, Savini G, Hammami S. Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021–2022. Viruses. 2024;16(3):362. https://doi.org/10.3390/v16030362. Boyer TC, Ward MP. Singer. Climate, Landscape, and the Risk of Orbivirus Exposure in Cattle in Illinois and Western Indiana. Am J Trop Med Hyg. 2010;83(4):789–94. 10.4269/ajtmh.2010.10-0132. Brand SPC, Keeling MJ. The impact of temperature changes on vector-borne disease transmission: Culicoides midges and bluetongue virus. J Royal Soc Interface. 2017;14:20160481. https://doi.org/10.1098/rsif.2016.0481. Chanda M, Carpenter S, Prasad G, et al. Livestock host composition rather than land use or climate explains spatial patterns in bluetongue disease in South India. Sci Rep. 2019;9:4229. https://doi.org/10.1038/s41598-019-40450-8. Chiuya T, Fèvre EM, Okumu NO, Abdi AM, Junglen S, Borgemeister C. Exposure to Arboviruses in Cattle: Seroprevalence of Rift Valley Fever, Bluetongue, and Epizootic Hemorrhagic Disease Viruses and Risk Factors in Baringo County, Kenya. Pathogens. 2024;13(8):613. https://doi.org/10.3390/pathogens13080613. Clavijo A, Sepulveda L, Riva J, Pessoa-Silva M, Tailor-Ruthes A, Lopez JW. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South America. Vet Rec. 2002;151:301–2. Ferrara G, Improda E, Piscopo F, Esposito R, Iovane G, Pagnini U, Montagnaro S. Bluetongue virus seroprevalence and risk factor analysis in cattle and water buffalo in southern Italy (Campania region). Vet Res Commun. 2024;48(1):579–84. 10.1007/s11259-023-10215-w. Epub 2023 Sep 8. PMID: 37682447; PMCID: PMC10810927. González-Recio O, Fernández A, Jiménez Montero JA. Epidemiological and genetic factors affecting severe epizootic hemorrhagic disease in Spanish Holstein cattle during the Southern Europe outbreak of 2023. J Dairy Sci. 2025;108(4):3850–7. 10.3168/jds.2024-25520. Epub 2025 Jan 7. PMID: 39788199. Gordon SJG, Bolwell C, Rogers CW, Musuka G, Kelly P, Guthrie A, Mellor PS, Hamblin C. A serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in Zimbabwe. Onderstepoort J Vet Res. 2017;84(1):e1–5. 10.4102/ojvr.v84i1.1505. PMID: 29227131; PMCID: PMC6238760. Groschupp S, Kampen H, Werner D. Winter activity of Culicoides (Diptera: Ceratopogonidae) inside and outside stables in Germany. Med Vet Entomol. 2024;38(3):552–65. https://doi.org/10.1111/mve.12756. Ibrahim A, Adam MA, Abdalla, Mohamed EH, Mohamed, Imadeldin E, Aradaib. Prevalence of bluetongue virus infection and associated risk factors among cattle in North Kordufan State, Western Sudan. BMC Veterinary Research 2014, 10:94 http://www.biomedcentral.com/1746-6148/10/94 Ishaq M, Jamal SM, Teodori L, Leone A, Bonfini B, Spedicato M, Savini G. Serological evidence of epizootic hemorrhagic disease and serotypes of epizootic hemorrhagic disease virus in Pakistan. Acta Trop. 2025;267:107675. 10.1016/j.actatropica.2025.107675. Epub 2025 May 25. PMID: 40425081. Jiménez-Cabello L, Utrilla-Trigo S, Lorenzo G, Ortego J, Calvo-Pinilla E. Epizootic Hemorrhagic Disease Virus: Current Knowledge and Emerging. Perspect Microorganisms. 2023;11(5):1339. 10.3390/microorganisms11051339. PMID: 37317313; PMCID: PMC10224379. Joardar SN. Prevalence and sero-epidemiology of bluetongue with special reference to eastern and north-eastern states of India. JoBAZ. 2022;83:9. https://doi.org/10.1186/s41936-022-00271-0. Kalthoum S, Sghaier S, Hassine B, Teodori T, Spedicato L, Guesmi M, Gharbi K, Hajlaoui R, Mohamed HBH, Khalfaoui B, Lachtar W, Ben Salem M, Fatnassi A, Dhaouadi N, Ben Ali A, Thabet M, Seghaier S, Savini C, G., Hammami S. Risk-based serological survey of bluetongue and the first evidence of bluetongue virus serotype 26 circulation in Tunisia. Veterinary Med Sci. 2022;8(4):1671–82. https://doi.org/10.1002/vms3.818. Kardjadj M, Luka PD, Benmahdi MH. Sero-epidemiology of bluetongue in Algerian ruminants. Afr J Biotechnol. 2016;15(20):868–71. https://doi.org/10.5897/AJB2016.15343. Lv MN, Zhu JB, Liao SQ, Yang ZX, Lin XH, Qi NS, Chen QL, Wu CY, Li J, Cai HM, Zhang JF, Hu JJ, Xiao WW, Zhang X, Sun MF. Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013–2017, China. Viruses. 2023;15(6):1263. 10.3390/v15061263. PMID: 37376563; PMCID: PMC10303234. Maclachlan NJ, Osburn BI. Epizootic haemorrhagic disease of deer. Infect Dis Livest. 2004;2(8):1227–30. Madani H, Casal J, Alba A, Allepuz A, Cêtre-Sossah C, Hafsi L, Kount-Chareb H, Bouayed-Chaouach N, Saadaoui H, Napp S. Animal diseases caused by orbiviruses, Algeria. Emerg Infect Dis. 2011;17:2325–7. https://doi.org/10.3201/eid1712.110928. Magliano A, Scaramozzino P, Ravagnan S, Montarsi F, DA Rold G, Cincinelli G, Moni A, Silvestri P, Carvelli A, DE Liberato C. Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy. Med Vet Entomol. 2018;32(1):70–7. 10.1111/mve.12260. Epub 2017 Aug 22. PMID: 28833269. Mahmoud A, Danzetta ML, di Sabatino D, Spedicato M, Alkhatal Z, Dayhum A, Tolari F, Forzan M, Mazzei M, Savini G. First seroprevalence investigation of epizootic haemorrhagic disease virus in Libya. Open Vet J 2021 Apr-Jun;11(2):301–8. doi: 10.5455/OVJ.2021.v11.i2.15. Epub 2021 Jun 21. PMID: 34307088; PMCID: PMC8288730. Manavian M, Hashemi M, Nikoo D, Tavan F, Hosseini SMH, Bakhshesh M, Marhamatizade MH. (2017) Seroprevalence of bluetongue virus infection and associated risk factors in domestic ruminants in the south of Iran, The Thai Journal of Veterinary Medicine , 47(2), pp. 225–231. available at: https://he01.tci-thaijo.org/index.php/tjvm/article/view/90280 Medrouh B, Abdelli A, Belkessa S, Ouinten Y, Brahimi M, Hakem A, Kernif T, Singer M, Ziam Tsaousis S, Jokelainen HAD, Savini P. Pasolli E.Seroprevalence and risk factors of bluetongue virus in domestic cattle, sheep, goats and camels in Africa: a systematic review and meta-analysis. Veterinary Q. 2024;44(1):1–12. https://doi.org/10.1080/01652176.2024.2396118. Mejri S, Dhaou SB, Jemli M, Breard E, Sailleau C et al. Epizootic haemorrhagic disease virus circulation in Tunisia. Veterinaria Italiana , 2018, 54 (1), pp.87–90. ff10.12834/VetIt.973.5129.2ff. ffhal-02621503f Navarro-Mamani DA, Jurado J, Vargas-Calla A, Ponce K, Sherman T, Zarate Y, Murga-Moreno CA, Perez I, Villacaqui R, Ara M, Ortiz P, Rivera H, Mayo CE. National Seroprevalence and Risk Factors of Bluetongue Virus in Domestic Ruminants of Peru. Transbound Emerg Dis. 2025;2025:2690231. 10.1155/tbed/2690231. PMID: 40302753; PMCID: PMC12016983. Neves dos Reis J, Suzan Varaschin M, Maria Seles Dorneles E, et al. Blue Tongue Virus in Dairy Cattle in the Southern Region of Minas Gerais, Brazil-Serological Survey. Acta Sci Veterinariae. 2022;50. https://doi.org/10.22456/1679-9216.127535. Noronha LE, Cohnstaedt LW, Richt JA, Wilson WC. Perspectives on the Changing Landscape of Epizootic Hemorrhagic Disease Virus Control. Viruses. 2021;13(11):2268. 10.3390/v13112268. PMID: 34835074; PMCID: PMC8618044. Orange JP, Dinh ETN, Goodfriend O, Citino SB, Wisely SM, Blackburn JK. Evidence of epizootic hemorrhagic disease virus and bluetongue virus exposure in nonnative ruminant species in northern Florida. J Zoo Wildl Med . 2021;51(4):745–751. 10.1638/2019-0174. PMID: 33480554. Purse BV, Mellor PS, Rogers DJ, Samuel AR, Mertens PPC, Baylis M. Climate change and the recent emergence of bluetongue in Europe. Nat Rev Microbiol. 2005;3(2):171–81. Ries C, Beer M, Hoffmann B. BlueTYPE—A low-density TaqMan-RT-qPCR array for the identification of all 24 classical Bluetongue virus serotypes. J Virol Methods. 2020;282:113881. https://doi.org/10.1016/j.jviromet.2020.113881. Ries C, Vogtlin A, Hüssy D, Jandt T, Gobet H, Hilbe M, Burgener C, Schweizer L, Häfliger-Speiser S, Beer M, Hoffmann B. Putative novel atypical BTV serotype 36 identified in small ruminants in Switzerland. Viruses. 2021;13(5):721. https://doi.org/10.3390/v13050721. Rivera NA, Varga C, Ruder MG, Dorak SJ, Roca AL, Novakofski JE, Mateus-Pinilla NE. Bluetongue and Epizootic Hemorrhagic Disease in the United States of America at the Wildlife–Livestock Interface. Pathogens 2021, 10, 915. https://doi.org/10.3390/pathogens10080915 Ross SM. (2014). Chapter 2 - Descriptive statistics. In S. M. Ross, editor, Introduction to probability and statistics for engineers and scientists (5th ed., pp. 9–51) Academic Press . https://doi.org/10.1016/B978-0-12-394811-3.50002-2 Sailleau C, Zanella G, Breard E, Viarouge C, Desprat A, Vitour D, Adam M, Lasne L, Martrenchar A, Bakkali-Kassimi L, Costes L, Zientara S. Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island. Vet Microbiol. 2012;155(2–4):191–7. 10.1016/j.vetmic.2011.09.006. Epub 2011 Sep 21. PMID: 22005178. Saminathan M, Singh KP, Khorajiya JH, Dinesh M, Vineetha S, Maity M, Rahman AF, Misri J, Malik YS, Gupta VK, Singh RK, Dhama K. An updated review on bluetongue virus: epidemiology, pathobiology, and advances in diagnosis and control with special reference to India. Vet Q. 2020;40(1):258–321. PMID: 33003985; PMCID: PMC7655031. Savini G, Afonso A, Mellor P, Aradaib I, Yadin H, Sanaa M, Wilson W, Monaco F, Domingo M. Epizootic heamorragic disease. Res Vet Sci. 2011;91(1):1–17. 10.1016/j.rvsc.2011.05.004. Epub 2011 Jun 12. PMID: 21665237. Schroeder ME, Johnson DJ, Ostlund EN, Meier J, Bounpheng MA, Clavijo A. Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of Bluetongue virus and Epizootic hemorrhagic disease virus. J Vet Diagn Invest. 2013;25(6):709–19. 10.1177/1040638713503654. Sghaier S, Sailleau C, Marcacci M, Thabet S, Curini V, Ben Hassine T, Teodori L, Portanti O, Hammami S, Jurisic L et al. Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021. Viruses 2023, 15 , 16. Şevik M. 2022. Seroprevalence of the Epizootic Hemorrhagic Disease Virus in cattle in the Aegean region of Turkey. Paper presented at the ISPEC 9th International Conference on Agriculture, Animal Sciences and Rural Development, Burdur, Turkey. Silvia E, León Trinidad MS, Christian Barrantes Bravo PD, Shefferson Feijoo Narvasta ZE, Ethel Huamán Fuertes MS, Gustavo A, Trigoso ZE, Francys C, Sáenz MS, Hurley A, Quispe-Ccasa. Dr. Seroprevalence of reproductive and infectious diseases in cattle: the case of Madre de Dios in the Peruvian southeastern tropics. Am J Vet Res. doi.org/10.2460/ajvr.23.08.0177 Wilson AJ, Mellor PS. Bluetongue in Europe: past, present and future. Philosophical Trans Royal Soc B: Biological Sciences. 2009;364(1530):2669–81. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":148279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eStudy area and sampled farms\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/d1af202f7e647ef50122d038.jpeg"},{"id":91561959,"identity":"53251c79-6970-44c5-9345-9edb63665d54","added_by":"auto","created_at":"2025-09-17 18:50:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePrevalence of EHD and BT at the herd and animal level\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/44c6bcd37bcba5b5372d5376.png"},{"id":91561949,"identity":"67d977ac-a655-4bc9-9bde-768496b014ad","added_by":"auto","created_at":"2025-09-17 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governorate\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/860630d2602faf03649b5393.png"},{"id":91561951,"identity":"ebb990f9-caf1-482a-b599-8e21fcbdb004","added_by":"auto","created_at":"2025-09-17 18:50:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePotential risk factors associated with BT and EHD seropositivity among cattle in Northwestern Tunisia\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/0efa994da97a33fcf73fca59.png"},{"id":101151837,"identity":"a25aeb3d-91a7-4b5a-8e87-b93d2711e7e5","added_by":"auto","created_at":"2026-01-26 16:06:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1072148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/9e9ebd35-9d99-4da0-828b-0b69e4d02caf.pdf"},{"id":91562707,"identity":"d4062c80-691f-4f0d-b0a0-82c023d96007","added_by":"auto","created_at":"2025-09-17 18:58:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":815438,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS1QuestionnaireEnglishversion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7398755/v1/ad64e39f130b52c045b7342b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seroprevalence and risk factors: A comprehensive seroepidemiological study of epizootic hemorrhagic disease and bluetongue in Northwestern Tunisia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eOrbivirus\u003c/em\u003e comprises several significant viruses that exert a detrimental effect on animal health. Vector-borne diseases caused by viruses such as epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) primarily affect livestock and wildlife, leading to severe outbreaks and significant economic losses in agriculture. Both viruses belong to the family \u003cem\u003eReoviridae\u003c/em\u003e and share morphological and structural similarities (Maclachlan \u0026amp; Osburn, 2004). As with other \u003cem\u003eOrbivirus\u003c/em\u003e members, BTV and EHDV possess seven structural proteins (VP1 to VP7), forming two capsids. The VP2 protein, exposed on the surface of the virion, serves as the serotype-specific antigen (Wilson \u0026amp; Mellor, 2009).These diseases are recognized as notifiable by the World Organization for Animal Health (Jim\u0026eacute;nez-Cabello et al., 2023).\u003c/p\u003e\u003cp\u003eThe EHDV was detected for the first time in 1955 in North America (Mejri et al., 2018). According to literature, seven serotypes of EHDV have been identified, numbered 1\u0026ndash;2 and 4\u0026ndash;8 (Ben Hassine et al., 2024). BT was first recognized as a disease of cattle in 1933 (Joardar S.N, 2022). As of now, there have been 36 distinct serotypes of BTV identified. This includes 24 classical serotypes and 12 that are classified as atypical (Ries et al., 2020; Ries et al., 2021).\u003c/p\u003e\u003cp\u003eResearch has demonstrated how climate change exacerbates the distribution of such diseases, thereby enabling their emergence in regions where they were previously absent (Savini et al., 2011; Purse et al., 2005; Clavijo et al., 2002). For instance, the spread of BTV has been impacted by changing weather patterns affecting insect dynamics, thereby increasing the risk of outbreaks in susceptible animal populations (Brand et al., 2017). BTV and EHDV are transmitted by \u003cem\u003eCulicoides\u003c/em\u003e biting midges, and their co-circulation is considered a common phenomenon (Savini et al., 2011).\u003c/p\u003e\u003cp\u003eTunisia is endemic to both viruses. The first incursion of BTV occurred in 1999, since then, multiple serotypes, including BTV-1, BTV-2, BTV-3, BTV-4, BTV-Y and BTV-26, have been identified (Kalthoum et al., 2022). The first outbreak of EHDV was documented in 2006, resulting in significant economic losses for various cattle farms due to high mortality and morbidity. The Tunisian EHDV strains were classified as serotype 6 (Ben Dhaou et al., 2016). In 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia (Sghaier et al., 2023). Despite neighbouring countries like Algeria showing a link between EHDV and BTV prevalence with vector distribution (\u003cem\u003eCulicoides imicola\u003c/em\u003e) (Hafsa Madani et al., 2011), data on EHDV prevalence and risk factors in Tunisia remain scarce, although risk factors for BTV have been studied in the country and an overall seroprevalence of 40.1% was reported (Kalthoum et al., 2022).\u003c/p\u003e\u003cp\u003eThe present study aims to assess the prevalence and distribution of EHDV antibodies in organized farms in northwest Tunisia. By identifying risk factors, it intends to provide valuable insights into EHDV and BT transmission dynamics, contributing to effective disease management strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy area\u003c/h2\u003e\u003cp\u003eThe study was conducted from 25 April to 19 May 2023, in four governorates in the north-western Tunisia : Jendouba, Beja, Siliana, and Kef (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These governorates were further subdivided into 76 districts and 744 sectors, covering an area of 16,267 km2, i.e. 10% of the total area of Tunisia with a total population of 1,210,000 inhabitants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The study area is the rainiest region of the country, receiving more than 1,000 millimetres of rainfall per year. It is characterised by the presence of important watercourses and reservoirs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSapmling design and serum collection\u003c/h3\u003e\n\u003cp\u003eThe study population consisted of organized farms in the north-western region of Tunisia, comprising 31 cattle farms from both the private and state sectors, with a total population of 8,634 cattle. As this is the first serological survey targeting EHDV and BT in the region, all farms within the area were included in the study. The exhaustive list of animals across the 31 farms was provided by the regional veterinary services and the Office of Livestock and Pastures (OEP). No animals were euthanized or sacrificed during this study; only non-invasive blood sampling was conducted.\u003c/p\u003e\u003cp\u003eThe following parameters were used to calculate of the required sample size:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eAn expected prevalence of 25%, based on histroical outbreaks data.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eA desired absolute precision of 15%\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eA 5% risk of error (reference) .\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eA total of 484 cattle were initially required. A proportional sampling approach was used to determine the number of cattle to be sampled per farm, ensuring that the sample accurately reflected the distribution of animals across the farms. To guarantee representativeness of our sample, the cattle to be sampled were randomly selected using the sampling frame. Only animals over six months of age were included in the study to avoid potential interference with passive immunity. It is important to note that the study exclusively enrolled cattle that had not previously been vaccinated against BTV. As no EHD vaccines are commercially available worldwide, this study provided an opportunity to assess the natural prevalence of EHD in the absence of vaccination. During the study, two farms initially scheduled to participate declined, reducing the final sample size to 396 cattle distributed across 29 farms.\u003c/p\u003e\u003cp\u003eA 10 ml serum was collected from each animal, strictly adherence to animal welfare standards. Each sample was distinctly labeled using a predefined code accurate identification. All the samples were centrifuged to separate the serum and then, stored at -20\u0026deg;C. The stored samples were subsequently transported under controlled conditions to the Institute of Veterinary Research of Tunisia (IRVT) for serological analysis.\u003c/p\u003e\n\u003ch3\u003eData collection\u003c/h3\u003e\n\u003cp\u003eThe study was carried out between April and May 2023. Farms and animals data were collected using a structured questionnaire specifically developed for this study consisting of three main parts. The first part focused on farm-related information, including the farm name, geographical location, type of building and housing conditions, total number of cattle, and the history of BT-like diseases on the farm. The second part is dedicated to individual animals and collected details such as age, sex, and breed. The final section covered potential risk factors associated with exposure to the BTV and EHDV, such as the introduction of animals, the presence of stagnant water, and proximity to wetlands. An English version of the questionnaire is provided as Supplementary File S1. Data was collected using an application of the Kobotoolbox platform(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.kobotoolbox.org\" target=\"_blank\"\u003ewww.kobotoolbox.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.kobotoolbox.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor the environmental risk factors, data on temperature were extracted from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ogimet.com\u003c/span\u003e\u003cspan address=\"https://www.ogimet.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e for the period ranged between 30 march 2020 and 31 may 2023.\u003c/p\u003e\n\u003ch3\u003eDetection of antibodies against BTV and EHDV\u003c/h3\u003e\n\u003cp\u003eAll sera were tested using the competitive enzyme-linked immunosorbent assay (ELISA) for EHDV (ID Screen\u0026reg; EHDV Competition, IDVet, France) and the BTV ELISA to detect antibodies directed against the VP7 protein (c-ELISA) kit (ID SCREEN\u0026reg; Bluetongue Competition ELISA, IDVet, Grabels, France). Analyses were performed according to the manufacturer's instructions. Samples with an IP greater than 60% were considered positive, indicating the presence of specific antibodies against EHDV or BTV.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe extracted data was organized into an Excel spreadsheet. The age variable was transformed into categorical variable with three defined age classes: Class 1(6\u0026ndash;12 months), Class 2 (13\u0026ndash;24 months) and Class 3 (more than 24 months). Herd size was categorized into three classes using the quartile classification method (Ross, 2014).\u003c/p\u003e\u003cp\u003eThe seroprevalence of BTV and EHDV was estimated at both the herd and individual levels and the 95% confidence interval (CI) was calculated using the binomial exact method. The apparent herd prevalence was calculated as the proportion of positive herds among the total number of herds tested. The prevalence at the individual level was determined by dividing the number of positive animals by the total number of animals tested in that herd.\u003c/p\u003e\u003cp\u003eRaster for temperature was created using the interpolation method and values on farm location were extracted using Arcgis function \u0026ldquo;extract values to point\u0026rdquo;.\u003c/p\u003e\u003cp\u003eRaster for NDVI was extracted from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.copernicus.eu/en\u003c/span\u003e\u003cspan address=\"https://www.copernicus.eu/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e for the period between 30 april 2019 and 31 may 2023. Values on farm location were also extracted using Arcgis function \u0026ldquo;extract values to point\u0026rdquo;.\u003c/p\u003e\u003cp\u003eUnivarite and multivariable mixed-effects logistic regression were conducted to identify risk factors for EHD and BTV at herd and animal level. The status of EHD and BT infection was designated as the outcome variable. The explanatory variables considered for univariate and multivariate analysis, are as follows : history of BT-like diseases on the farm, type of building, distance from wetlands, presence of mosquitoes at the farm, presence of stagnant water on the farm, animal exchange with other farms, presence of wetlands close to the farm, age, sex, herd size, and breed. Associations between seroprevalence of EHD and BT and potential risk factors were initially assessed through univariable analysis and only variables with p\u0026thinsp;\u0026lt;\u0026thinsp;0.2 were subsequently included in the logistic multiple-regression model. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated. The presence of multicollinearity among the variables was evaluated using generalized variance inflation factors (GVIFs). The Hosmer-Lemeshow goodness-of-fit test was performed on the final model. Furthermore, receiver operating characteristic (ROC) curves and area under the curve (AUC) values were calculated in order to assess the model\u0026rsquo;s predictive performance.\u003c/p\u003e\u003cp\u003eAll statistical analyses were performed using R software, and the maps were generated by ArcGIS version 10.4.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eCattle and farm characteristics\u003c/h2\u003e\u003cp\u003eA total of 394 cattle, consisting of 386 females (97.9%) and 8 males (2.03%) belonging to 29 farms in four north-western governorates, were recruited for this study. As for the demographic characteristics of the sample, the age of the cattle ranged from 7 to 123 months with a mean of 40 months. The breed of the sample was predominantly Holstein constituting 85.02% (335/394) of the total. Other breeds including Montbeliarde, Swiss and Tarentaise accounted for 14.84% (59/394). For the gender, the sample is mainly of females accounting for 97.9% (386 out of 394). In term of age, the animals are categorised into three distinct age classes: 6\u0026ndash;12 months, 13\u0026ndash;24 months and over 25 months. The age group most represented is that of cattle over 25 months of age, representing 67.5% (266/394) of the sampled animals. The characteristics of the surveyd farms and cattle are presented in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristic of the surveyed famrs and sampled animals\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFrequency\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHerd size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLarge [\u0026gt;\u0026thinsp;300]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 (17.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSmall [\u0026lt;\u0026thinsp;300]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24 (82.7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge class\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31 (7.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e97 (24.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[25\u0026ndash;123]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e266 (67.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e386 (97.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (2,03%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBreed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHolstein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e335 (80.02%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMontbeliarde\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (3.04%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSwiss\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37 (9.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTarentaise\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (2.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHousing type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIndoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (2.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSemi-enclosed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e384 (97.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSeroprevalence of the EHD and BT\u003c/h3\u003e\n\u003cp\u003eAt the herd level, all the farms were tested positive for the BT (29/29 (100%)) while 93.1% (95% CI: 77.23\u0026ndash;99.15%) (27/29) were tested positive for anti-EHDV antibodies. At the individual animal level, 198 out of 394 samples tested positive for EHD giving an overall seroprevalence rate of 50.3% (95% CI: 45.2%-55.3%). For BT, 81,2% (320/394) (95% CI: 77%-85%) of the tested animals were positive (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrevalence of EHDV and BT at the herd and animal level\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDisease\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHerd prevalence (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAnimal prevalence (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEHDV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e93.1% [77.2\u0026ndash;99.2%]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50.3% [45.2\u0026ndash;55.3%]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100% [88.1\u0026ndash;100%]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e81.2% [77\u0026ndash;85%]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe presence of dual exposure to BT and EHD was detected in 27 (93.1%) of the tested herds. At the individual level, dual exposure to BT and EHD was detected in 182 (46.2%) of the total number of cattle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAccording to the governorate, 7 cattle out of 10 selected from the Kef governorate, were found to be positive to EHD representing the highest level of the seropositivity followed by Jendouba where 54.8% (91/166) of the cattle tested were positive. The lowest seroprevalence of EHD was recorded in Beja with 44.2% (61/138) of the cattle being seropositive. However, statistical analysis revealed no statistically significant differences between the governorates (p-value\u0026thinsp;=\u0026thinsp;0.169). However, for BT, the results indicate that Siliana has the highest prevalence of BT with 92.5% (74/80), followed by Jendouba (82.5% (137/166), Beja (76.1% (105/138) and Kef (40% (4/10)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The observed variation in the prevalence of BT across governorates was found to be statistically significant (p-value\u0026thinsp;=\u0026thinsp;0.00000).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRisk factors of EHD and BT\u003c/h2\u003e\u003cp\u003eUnivariate analysis revealed that the age and temperature were identified as significant predictors of BT. As demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the risk factors associated with a positive test result for EHD were distance from wetlands, age and the presence of wetlands (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The final model demonstrated that the age was the only significant predictor for both EHD and BT. Animals aged 25\u0026ndash;123 months (Category 3) have significantly higher odds of seropositivity of BT compared to younger (OR\u0026thinsp;=\u0026thinsp;5.30, 95% CI: 1.77\u0026ndash;15.87, p\u0026thinsp;=\u0026thinsp;0.002). While temperature showed a non-significant association with BT seropositivity (OR\u0026thinsp;=\u0026thinsp;1.57, 95% CI: 0.91\u0026ndash;2.73, p\u0026thinsp;=\u0026thinsp;0.10). Regarding EHD, both category of age (Category 2 and 3) demonstrated significantly increased odds compared to younger animals (Category 2 :12\u0026ndash;24 months; OR\u0026thinsp;=\u0026thinsp;7.81, 95% CI: 1.54\u0026ndash;39.52, p\u0026thinsp;=\u0026thinsp;0.01, Category 3 :25\u0026ndash;123 months; OR\u0026thinsp;=\u0026thinsp;21.1, 95% CI: 4.38-102.11, p\u0026thinsp;=\u0026thinsp;0.0000). Farms located less then 1 km from wetlands tended to have lower odds of EHD seropositivity, although, this association did not reach statistical significance (OR\u0026thinsp;=\u0026thinsp;0.25, 95% CI: 0.059\u0026ndash;1.11, p\u0026thinsp;=\u0026thinsp;0.07) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All other variables, including housing type, breed, mosquito presence in the farm, NDVI, stagnant water, and flock size, showed no significant associations with either disease in the univariate analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eUnivariate analysis for the association between potential risk factors and BT and EHD seropositivity among cattle in Northwestern Tunisia\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCategory\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eEHD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eOR [IC95%]\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eOR [IC95%]\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHousing type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSemi-enclosed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.44 [0.06\u0026ndash;34.42]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.62 [0.09\u0026ndash;28.14]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBreed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHolstein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.63 [0.15\u0026ndash;2.70]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.95 [0.29\u0026ndash;3.09]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistacne from wetlands\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDistance\u0026thinsp;\u0026lt;\u0026thinsp;1 km\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.59 [0.17\u0026ndash;2.05]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.26 [0.06\u0026ndash;1.02]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresence of mosquitos in the farm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.51 [0.07\u0026ndash;3.74]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.81 [0.15\u0026ndash;4.36]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDVI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNDVI (per unit)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.07 [0.05\u0026ndash;22.49]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.85 [0.06\u0026ndash;11.08]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresence of stagnant water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.46 [0.15\u0026ndash;14.53]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.03 [0.13\u0026ndash;8.20]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAge class\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory 2 : [12\u0026ndash;24 moths]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.02 [0.33\u0026ndash;3.14]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.02 [1.98\u0026ndash;24.87]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory 3 : [25\u0026ndash;123 months]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.31 [1.77\u0026ndash;15.87]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.20 [5.91\u0026ndash;69.03]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlock size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[\u0026gt;\u0026thinsp;300]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.72 [0.20\u0026ndash;2.54]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.92 [0.30\u0026ndash;2.78]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature (per unit)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.52 [0.94\u0026ndash;2.47]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.09\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.25 [0.79\u0026ndash;1.97]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMultivariate analysis for the association between potential risk factors and BT and EHD seropositivity among cattle in Northwestern Tunisia\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCategory\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eBT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eEHD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOR [IC95%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOR [IC95%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistacne from wetlands\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDistance\u0026thinsp;\u0026lt;\u0026thinsp;1 km\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25 [0.059\u0026ndash;1.11]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAge class\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory 2 : [12\u0026ndash;24 moths]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.81 [1.54\u0026ndash;39.52]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory 3 : [25\u0026ndash;123 months]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.30 [1.77\u0026ndash;15.87]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.16 [4.38-102.11]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature (per unit)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.57 [0.91\u0026ndash;2.73]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn Tunisia, the surveillance system of vector-borne diseases such as BT and EHD relies only on the passive surveillance. Since the first report of EHD in Tunisia and according to the previous study, the northwestern region has been identified as a highly infected area. Both diseases (BT and EHD) can be transmitted by the same vector and cases of co-infection have been previously documented (Ben Hassine et al., 2024). To our knowledge, few studies have been considered the seroprevalence of the vector-borne diseases specially in the northwestern Tunisia. Therefore, the present study aims to assess the serporevalence of BT and EHD in organized farms in the northwestern Tunisia and to identify potential risk factors.\u003c/p\u003e\u003cp\u003eThe results of this study indicate the widespread of the BT and EHD in the northern Tunisia. The overall seroprevalence of EHD estimated animal-level in this study (50.3%) is considerably higher than that reported in previous studies. Mejri et al. (2018), reported a seroprevalence of 8.6% during a sentinel study conducted in cattle between 2013 and 2014, Ben Dhaou (2017) detected a seroprevalence of 5.3% in the governorate of Bizerte in 2012. This notable increase in seroprevalence may reflect a recent intensification in virus circulation and can be attributed to several factors such as favorable climatic conditions for vector activity and changes in vector competences, abundance and distribution (Gonz\u0026aacute;lez-Recio at al.2025). It was demonstrated that significant changes have been observed in the pattern of disease and distribution of EHDV and an increase in the severity of the disease among bovine populations (Noronha et al., 2021; Jim\u0026eacute;nez-Cabello et al., 2023). Our results align with the findings of various authors across different countries : 57.87% in China, 57.1% USA (Northern Florida), 66.9% in Pakistan and 64% in Kenya (Gordon et al., 2017; Orange et al., 2021; Lv et al., 2023; Ishaq et al., 2025). In contrast, studies conducted in neighbouring and non-neighbouring countries reported a low seroprevalences of 1% and 9% in cattle in Libya in 2021, Algeria in 2011 and India in 2010, respectively (Boyer et al., 2010; Madani et al., 2011; Mahmoud et al., 2021 ).\u003c/p\u003e\u003cp\u003eFor BT, the overall seroprevalence at the individual level was very high (81.2%) in our study indicating a high degree of exposure of cattle in the northern Tunisia to the virus. This result suggests recent circulation of the virus of BT in the study area. The observed high seroprevalence in these farms is likely indicative of a substantial population of Culicoides vectors that is facilitated by favorable climatic conditions. As demonstrated in the relevant literature, comparable high seroprevalences of BT have been reported in Kenya (91.5%), Estern Sudan (92.9%) and Brazil (83.3%) (Chiuya et al., 2024; Abraheem et al., 2024; Neves et al., 2022). However, the seroprevalences of BT reported in southern Italy (43.6%), Iran (27.63%), Algeria (13.7%) and Peru (19.3%) were lower than our finding (Kardjadj et al., 2016; Ferrara et al., 2024, Bakhshesh et al., 2020; Navarro-Mamani et al., 2025).\u003c/p\u003e\u003cp\u003eThe present study indicated that 46.2% of the tested cattle were co-infected with both BT and EHD. This is considered a high prevalence in comparison to the studies conducted in Mayotte and USA, where 9.4% and 1.49% of the samples were positive for both diseases, respectively (Schroeder et al., 2013; Sailleau et al., 2012). However, it aligns with the findings of Min-Na Lv et al. in China in 2012\u0026ndash;2013 (40.3%) (Lv et al., 2023). The presence of BT and EHD coinfections observed in our study can be attributed to several factors. The viruses that cause theses diseases are both transmitted by the same genus of hematophagous biting midges (Culicoides spp.) and when the ecological niches of the two diseases overlap (Lv et al., 2023). Consistent with other research (Boyer et al., 2010; Rivera et al., 2021), our results confirm that significant exposure to EHDV and BTV is associated with the abundance of Culicoides spp. in the region.\u003c/p\u003e\u003cp\u003eThe study revealed no significant differences in the EHD seroprevalence across governorates suggesting uniform exposure of cattle to this disease in the study area, wich may be due to similar environmental conditions and vector abundance. However, a significant variation in the seroprevalence of BT was highlighted, wich can be attributed to the differences in livestock species composition, breed susceptibility, and host and vector density (Chanda et al., 2019; Saminathan et al., 2020). Additionnaly, the seroprevalence of BT may be influenced by herd management practices, including vector control measures and grazing patterns (Beyen el al., 2025). C\u003c/p\u003e\u003cp\u003eBased on the univariate and multivariate analysis, we studied the association between seroprevalence of BT and EHD and the following risks factors ; Housing type, breed, distacne from wetlands, presence of mosquitos in the farm, NDVI, presence of stagnant water, age class, flock size and temperature. The results revealed that only the age is a risk factor for both diseases in the final model, but with stronger effects observed for EHD, reflecting distinct epidemiological dynamics between the two infections. The age-related increase in seroprevalence observed for both viruses aligns with findings from previous studies, which attribute higher exposure over time to the cumulative effect of vector contact as cattle age (Ibrahim et al., 2014; Bachir Medrouh et al., 2024; Chiuya et al., 2024; Şevik M, 2022). This highlights the endemic nature of these pathogens in environments with a high prevalence of vectors.\u003c/p\u003e\u003cp\u003eThe absence of significant breed-specific differences in seroprevalence of the two vector-borne diseases, previous studies conducted overwide suggest that susceptibility to EHD and BT in endemic areas is primarily determined by vector exposure rather than genetic factors (Manavian et al., 2017; Silvia et al., 2023). Furthermore, the seroprevalence of both diseases was found not to be associated with housing type. This finding is not unexpected, given that Culicoides are indoor and outdoor vectors and seasonal variation in climatic conditions influences their indoor and outdoor activity patterns (Magliano et al., 2018; Groschupp et al., 2024). The other investiguated risk factors were found to be significantly associated with either disease.\u003c/p\u003e\u003cp\u003eDespite the valuable data provided by this study, several limitations should be acknowledged. First, the study relied on cross-sectional serological data, which limits the ability to ascertain whether the viruses under investigation are recent or old circulation. Furthermore, environmental data used in this study such as temperature and NDVI were extracted from external sources and did not cover all the time periods of exposure, which may influence the association of these variables with BT and EHD seroprevalecne. Finally, the study did not include molecular detection of active infections or virus typing, which could provide deeper insights into circulating strains and the dynamics of co-infections.\u003c/p\u003e\u003cp\u003eThe findings of the present study enhance our comprehension of the epidemiological profiles of two vector-borne diseases (BT and EHD) in northern Tunisia, as well as the risk factors associated with their seroprevalences. The study provides valuable information for the control programmes of these diseases and for the adaptation of strategies in order to reduce the risk of their occurrence.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study highlights the widespread of BT and EHD in organised cattle farms across the northwesterern Tunisia. The high seroprevalence of both diseases indicates favourable environment for the vectors implicated in their transmission and spread. Although the study provided valuable data on the seroprevalence of the two diseases, it remains unclear whether these infections were occured. Future longitudinal studies incorporating molecular diagnostics and entomological surveillance are needed to better understand the transmission dynamics and temporal trends of BT and EHD in Tunisia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the veterinarians and the staff of the organized farms, whose support made this work possible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImed Ben Slimen, Sana Kalthoum, Kaouther Guesmi, Mohamed Naceur Baccar : \u0026nbsp;Conceptualization and methodology , formal analysis, writing-review and editing\u003c/p\u003e\n\u003cp\u003eImed Ben Slimen : \u0026nbsp; writing-original draft preparation\u003c/p\u003e\n\u003cp\u003eAida Tlatli, Soufien Sghaier, Aida Megdich, Sonia Ben Hsan, Salma Hadouchi : Laboratory analysis\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Hanen Ncibi, Chafik Ben Salah, Ilyes Arfaoui, Mohamed Yahya \u0026nbsp;Dalhoumi, \u0026nbsp;Marwa Sallami, Raja Gharbi: investigation and data collection \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval ans consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the \u003cstrong\u003eAnimalEthicsCommittee-NationalSchool of Veterinary Medicine,SidiThabet\u0026nbsp;\u003c/strong\u003e under the approval number\u003cstrong\u003e\u0026nbsp; CEEA-ENMV92/25\u003c/strong\u003e. Informed consent was obtained verbally from all participating animal owners after explaining the objectives of the study, and participation was entirely voluntary. No animals were euthanized or sacrificed during this study; only non-invasive blood sampling was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbraheem HH, Hussien MO, Elhassan AM, Enan KA, Musa AB, Ahmed SK, El Hussein ARM. Seroepidemiological Survey on Bluetongue Virus (BTV) among Cattle, Sheep, and Goats in Gadarif State, Eastern Sudan. Int J Microbiol. 2024;2024:7712412. 10.1155/2024/7712412. PMID: 39328248; PMCID: PMC11424855.\u003c/li\u003e\n\u003cli\u003eBakhshesh M, Otarod V, Fallah Mehrabadi MH. Large-scale seroprevalence and risk factors associated with Bluetongue virus in Iran. Prev Vet Med. 2020;179:104994. 10.1016/j.prevetmed.2020.104994. Epub 2020 Apr 30. PMID: 32402914.\u003c/li\u003e\n\u003cli\u003eBen Dhaou S. Etude de la maladie épizootique hémorragique en Tunisie. Sciences agricoles. Université Paris-Est; Faculté des sciences de Bizerte (Tunisie), 2017. Français. ffNNT: 2017PESC0023ff. fftel-01763049f.\u003c/li\u003e\n\u003cli\u003eBen Dhaou S, Sailleau C, Babay B, Viarouge C, Sghaier S, Zientara S, Hammami S, Bréard E. Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006. \u003cem\u003eActa Vet Hung\u003c/em\u003e. 2016;64(2):250 – 62. 10.1556/004.2016.025. PMID: 27342096.\u003c/li\u003e\n\u003cli\u003eBen Hassine T, García-Carrasco J-M, Sghaier S, Thabet S, Lorusso A, Savini G, Hammami S. Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021–2022. Viruses. 2024;16(3):362. https://doi.org/10.3390/v16030362.\u003c/li\u003e\n\u003cli\u003eBoyer TC, Ward MP. Singer. Climate, Landscape, and the Risk of Orbivirus Exposure in Cattle in Illinois and Western Indiana. Am J Trop Med Hyg. 2010;83(4):789–94. 10.4269/ajtmh.2010.10-0132.\u003c/li\u003e\n\u003cli\u003eBrand SPC, Keeling MJ. The impact of temperature changes on vector-borne disease transmission: Culicoides midges and bluetongue virus. J Royal Soc Interface. 2017;14:20160481. https://doi.org/10.1098/rsif.2016.0481.\u003c/li\u003e\n\u003cli\u003eChanda M, Carpenter S, Prasad G, et al. Livestock host composition rather than land use or climate explains spatial patterns in bluetongue disease in South India. Sci Rep. 2019;9:4229. https://doi.org/10.1038/s41598-019-40450-8.\u003c/li\u003e\n\u003cli\u003eChiuya T, Fèvre EM, Okumu NO, Abdi AM, Junglen S, Borgemeister C. Exposure to Arboviruses in Cattle: Seroprevalence of Rift Valley Fever, Bluetongue, and Epizootic Hemorrhagic Disease Viruses and Risk Factors in Baringo County, Kenya. Pathogens. 2024;13(8):613. https://doi.org/10.3390/pathogens13080613.\u003c/li\u003e\n\u003cli\u003eClavijo A, Sepulveda L, Riva J, Pessoa-Silva M, Tailor-Ruthes A, Lopez JW. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South America. Vet Rec. 2002;151:301–2.\u003c/li\u003e\n\u003cli\u003eFerrara G, Improda E, Piscopo F, Esposito R, Iovane G, Pagnini U, Montagnaro S. Bluetongue virus seroprevalence and risk factor analysis in cattle and water buffalo in southern Italy (Campania region). Vet Res Commun. 2024;48(1):579–84. 10.1007/s11259-023-10215-w. Epub 2023 Sep 8. PMID: 37682447; PMCID: PMC10810927.\u003c/li\u003e\n\u003cli\u003eGonzález-Recio O, Fernández A, Jiménez Montero JA. Epidemiological and genetic factors affecting severe epizootic hemorrhagic disease in Spanish Holstein cattle during the Southern Europe outbreak of 2023. J Dairy Sci. 2025;108(4):3850–7. 10.3168/jds.2024-25520. Epub 2025 Jan 7. PMID: 39788199.\u003c/li\u003e\n\u003cli\u003eGordon SJG, Bolwell C, Rogers CW, Musuka G, Kelly P, Guthrie A, Mellor PS, Hamblin C. A serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in Zimbabwe. Onderstepoort J Vet Res. 2017;84(1):e1–5. 10.4102/ojvr.v84i1.1505. PMID: 29227131; PMCID: PMC6238760.\u003c/li\u003e\n\u003cli\u003eGroschupp S, Kampen H, Werner D. Winter activity of Culicoides (Diptera: Ceratopogonidae) inside and outside stables in Germany. Med Vet Entomol. 2024;38(3):552–65. https://doi.org/10.1111/mve.12756.\u003c/li\u003e\n\u003cli\u003eIbrahim A, Adam MA, Abdalla, Mohamed EH, Mohamed, Imadeldin E, Aradaib. Prevalence of bluetongue virus infection and associated risk factors among cattle in North Kordufan State, Western Sudan. \u003cem\u003eBMC Veterinary Research\u003c/em\u003e 2014, 10:94 http://www.biomedcentral.com/1746-6148/10/94\u003c/li\u003e\n\u003cli\u003eIshaq M, Jamal SM, Teodori L, Leone A, Bonfini B, Spedicato M, Savini G. Serological evidence of epizootic hemorrhagic disease and serotypes of epizootic hemorrhagic disease virus in Pakistan. Acta Trop. 2025;267:107675. 10.1016/j.actatropica.2025.107675. Epub 2025 May 25. PMID: 40425081.\u003c/li\u003e\n\u003cli\u003eJiménez-Cabello L, Utrilla-Trigo S, Lorenzo G, Ortego J, Calvo-Pinilla E. Epizootic Hemorrhagic Disease Virus: Current Knowledge and Emerging. Perspect Microorganisms. 2023;11(5):1339. 10.3390/microorganisms11051339. PMID: 37317313; PMCID: PMC10224379.\u003c/li\u003e\n\u003cli\u003eJoardar SN. Prevalence and sero-epidemiology of bluetongue with special reference to eastern and north-eastern states of India. JoBAZ. 2022;83:9. https://doi.org/10.1186/s41936-022-00271-0.\u003c/li\u003e\n\u003cli\u003eKalthoum S, Sghaier S, Hassine B, Teodori T, Spedicato L, Guesmi M, Gharbi K, Hajlaoui R, Mohamed HBH, Khalfaoui B, Lachtar W, Ben Salem M, Fatnassi A, Dhaouadi N, Ben Ali A, Thabet M, Seghaier S, Savini C, G., Hammami S. Risk-based serological survey of bluetongue and the first evidence of bluetongue virus serotype 26 circulation in Tunisia. Veterinary Med Sci. 2022;8(4):1671–82. https://doi.org/10.1002/vms3.818.\u003c/li\u003e\n\u003cli\u003eKardjadj M, Luka PD, Benmahdi MH. Sero-epidemiology of bluetongue in Algerian ruminants. Afr J Biotechnol. 2016;15(20):868–71. https://doi.org/10.5897/AJB2016.15343.\u003c/li\u003e\n\u003cli\u003eLv MN, Zhu JB, Liao SQ, Yang ZX, Lin XH, Qi NS, Chen QL, Wu CY, Li J, Cai HM, Zhang JF, Hu JJ, Xiao WW, Zhang X, Sun MF. Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013–2017, China. Viruses. 2023;15(6):1263. 10.3390/v15061263. PMID: 37376563; PMCID: PMC10303234.\u003c/li\u003e\n\u003cli\u003eMaclachlan NJ, Osburn BI. Epizootic haemorrhagic disease of deer. Infect Dis Livest. 2004;2(8):1227–30.\u003c/li\u003e\n\u003cli\u003eMadani H, Casal J, Alba A, Allepuz A, Cêtre-Sossah C, Hafsi L, Kount-Chareb H, Bouayed-Chaouach N, Saadaoui H, Napp S. Animal diseases caused by orbiviruses, Algeria. Emerg Infect Dis. 2011;17:2325–7. https://doi.org/10.3201/eid1712.110928.\u003c/li\u003e\n\u003cli\u003eMagliano A, Scaramozzino P, Ravagnan S, Montarsi F, DA Rold G, Cincinelli G, Moni A, Silvestri P, Carvelli A, DE Liberato C. Indoor and outdoor winter activity of Culicoides biting midges, vectors of bluetongue virus, in Italy. Med Vet Entomol. 2018;32(1):70–7. 10.1111/mve.12260. Epub 2017 Aug 22. PMID: 28833269.\u003c/li\u003e\n\u003cli\u003eMahmoud A, Danzetta ML, di Sabatino D, Spedicato M, Alkhatal Z, Dayhum A, Tolari F, Forzan M, Mazzei M, Savini G. First seroprevalence investigation of epizootic haemorrhagic disease virus in Libya. Open Vet J 2021 Apr-Jun;11(2):301–8. doi: 10.5455/OVJ.2021.v11.i2.15. Epub 2021 Jun 21. PMID: 34307088; PMCID: PMC8288730.\u003c/li\u003e\n\u003cli\u003eManavian M, Hashemi M, Nikoo D, Tavan F, Hosseini SMH, Bakhshesh M, Marhamatizade MH. (2017) Seroprevalence of bluetongue virus infection and associated risk factors in domestic ruminants in the south of Iran, \u003cem\u003eThe Thai Journal of Veterinary Medicine\u003c/em\u003e, 47(2), pp. 225–231. available at: https://he01.tci-thaijo.org/index.php/tjvm/article/view/90280\u003c/li\u003e\n\u003cli\u003eMedrouh B, Abdelli A, Belkessa S, Ouinten Y, Brahimi M, Hakem A, Kernif T, Singer M, Ziam Tsaousis S, Jokelainen HAD, Savini P. Pasolli E.Seroprevalence and risk factors of bluetongue virus in domestic cattle, sheep, goats and camels in Africa: a systematic review and meta-analysis. Veterinary Q. 2024;44(1):1–12. https://doi.org/10.1080/01652176.2024.2396118.\u003c/li\u003e\n\u003cli\u003eMejri S, Dhaou SB, Jemli M, Breard E, Sailleau C et al. Epizootic haemorrhagic disease virus circulation in Tunisia. \u003cem\u003eVeterinaria Italiana\u003c/em\u003e, 2018, 54 (1), pp.87–90. ff10.12834/VetIt.973.5129.2ff. ffhal-02621503f\u003c/li\u003e\n\u003cli\u003eNavarro-Mamani DA, Jurado J, Vargas-Calla A, Ponce K, Sherman T, Zarate Y, Murga-Moreno CA, Perez I, Villacaqui R, Ara M, Ortiz P, Rivera H, Mayo CE. National Seroprevalence and Risk Factors of Bluetongue Virus in Domestic Ruminants of Peru. Transbound Emerg Dis. 2025;2025:2690231. 10.1155/tbed/2690231. PMID: 40302753; PMCID: PMC12016983.\u003c/li\u003e\n\u003cli\u003eNeves dos Reis J, Suzan Varaschin M, Maria Seles Dorneles E, et al. Blue Tongue Virus in Dairy Cattle in the Southern Region of Minas Gerais, Brazil-Serological Survey. Acta Sci Veterinariae. 2022;50. https://doi.org/10.22456/1679-9216.127535.\u003c/li\u003e\n\u003cli\u003eNoronha LE, Cohnstaedt LW, Richt JA, Wilson WC. Perspectives on the Changing Landscape of Epizootic Hemorrhagic Disease Virus Control. Viruses. 2021;13(11):2268. 10.3390/v13112268. PMID: 34835074; PMCID: PMC8618044.\u003c/li\u003e\n\u003cli\u003eOrange JP, Dinh ETN, Goodfriend O, Citino SB, Wisely SM, Blackburn JK. Evidence of epizootic hemorrhagic disease virus and bluetongue virus exposure in nonnative ruminant species in northern Florida. \u003cem\u003eJ Zoo Wildl Med\u003c/em\u003e. 2021;51(4):745–751. 10.1638/2019-0174. PMID: 33480554.\u003c/li\u003e\n\u003cli\u003ePurse BV, Mellor PS, Rogers DJ, Samuel AR, Mertens PPC, Baylis M. Climate change and the recent emergence of bluetongue in Europe. Nat Rev Microbiol. 2005;3(2):171–81.\u003c/li\u003e\n\u003cli\u003eRies C, Beer M, Hoffmann B. BlueTYPE—A low-density TaqMan-RT-qPCR array for the identification of all 24 classical Bluetongue virus serotypes. J Virol Methods. 2020;282:113881. https://doi.org/10.1016/j.jviromet.2020.113881.\u003c/li\u003e\n\u003cli\u003eRies C, Vogtlin A, Hüssy D, Jandt T, Gobet H, Hilbe M, Burgener C, Schweizer L, Häfliger-Speiser S, Beer M, Hoffmann B. Putative novel atypical BTV serotype 36 identified in small ruminants in Switzerland. Viruses. 2021;13(5):721. https://doi.org/10.3390/v13050721.\u003c/li\u003e\n\u003cli\u003eRivera NA, Varga C, Ruder MG, Dorak SJ, Roca AL, Novakofski JE, Mateus-Pinilla NE. Bluetongue and Epizootic Hemorrhagic Disease in the United States of America at the Wildlife–Livestock Interface. \u003cem\u003ePathogens\u003c/em\u003e 2021, 10, 915. https://doi.org/10.3390/pathogens10080915\u003c/li\u003e\n\u003cli\u003eRoss SM. (2014). Chapter 2 - Descriptive statistics. In S. M. Ross, editor, Introduction to probability and statistics for engineers and scientists (5th ed., pp. 9–51) \u003cem\u003eAcademic Press\u003c/em\u003e. https://doi.org/10.1016/B978-0-12-394811-3.50002-2\u003c/li\u003e\n\u003cli\u003eSailleau C, Zanella G, Breard E, Viarouge C, Desprat A, Vitour D, Adam M, Lasne L, Martrenchar A, Bakkali-Kassimi L, Costes L, Zientara S. Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island. Vet Microbiol. 2012;155(2–4):191–7. 10.1016/j.vetmic.2011.09.006. Epub 2011 Sep 21. PMID: 22005178.\u003c/li\u003e\n\u003cli\u003eSaminathan M, Singh KP, Khorajiya JH, Dinesh M, Vineetha S, Maity M, Rahman AF, Misri J, Malik YS, Gupta VK, Singh RK, Dhama K. An updated review on bluetongue virus: epidemiology, pathobiology, and advances in diagnosis and control with special reference to India. Vet Q. 2020;40(1):258–321. PMID: 33003985; PMCID: PMC7655031.\u003c/li\u003e\n\u003cli\u003eSavini G, Afonso A, Mellor P, Aradaib I, Yadin H, Sanaa M, Wilson W, Monaco F, Domingo M. Epizootic heamorragic disease. Res Vet Sci. 2011;91(1):1–17. 10.1016/j.rvsc.2011.05.004. Epub 2011 Jun 12. PMID: 21665237.\u003c/li\u003e\n\u003cli\u003eSchroeder ME, Johnson DJ, Ostlund EN, Meier J, Bounpheng MA, Clavijo A. Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of Bluetongue virus and Epizootic hemorrhagic disease virus. J Vet Diagn Invest. 2013;25(6):709–19. 10.1177/1040638713503654.\u003c/li\u003e\n\u003cli\u003eSghaier S, Sailleau C, Marcacci M, Thabet S, Curini V, Ben Hassine T, Teodori L, Portanti O, Hammami S, Jurisic L et al. Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021. \u003cem\u003eViruses\u003c/em\u003e 2023, \u003cem\u003e15\u003c/em\u003e, 16.\u003c/li\u003e\n\u003cli\u003eŞevik M. 2022. Seroprevalence of the Epizootic Hemorrhagic Disease Virus in cattle in the Aegean region of Turkey. Paper presented at the ISPEC 9th International Conference on Agriculture, Animal Sciences and Rural Development, Burdur, Turkey.\u003c/li\u003e\n\u003cli\u003eSilvia E, León Trinidad MS, Christian Barrantes Bravo PD, Shefferson Feijoo Narvasta ZE, Ethel Huamán Fuertes MS, Gustavo A, Trigoso ZE, Francys C, Sáenz MS, Hurley A, Quispe-Ccasa. Dr. Seroprevalence of reproductive and infectious diseases in cattle: the case of Madre de Dios in the Peruvian southeastern tropics. Am J Vet Res. doi.org/10.2460/ajvr.23.08.0177\u003c/li\u003e\n\u003cli\u003eWilson AJ, Mellor PS. Bluetongue in Europe: past, present and future. Philosophical Trans Royal Soc B: Biological Sciences. 2009;364(1530):2669–81.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Epizootic hemorrhagic disease, Bluetongue, seroprevalence, survey, risk factors, cattle, Tunisia","lastPublishedDoi":"10.21203/rs.3.rs-7398755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7398755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpizootic hemorrhagic disease (EHD) and Bluetongue (BT) are vector-borne diseases that commonly circulate among wild and domestic ruminants. The epidemiological situation of EHD in Tunisia remains poorly documented, despite several studies having been conducted on BT. To assess the seroprevalence of the EHD and BT in northern Tunisia, 394 bovine serum samples were collected and tested for anti-VP2 antibodies using a competitive ELISA. The seroprevalence of EHD and BT at the individual level was estimated at 51.2% and 81% respectively. Herd-level seroprevalence reached 93% for EHDV and 100% for BTV. No statistically significant differences in prevalence were observed between governorates for EHD (p-value\u0026thinsp;=\u0026thinsp;0.169). However, the prevalence of BT across governorates was found to be statistically significant (p-value\u0026thinsp;=\u0026thinsp;0.00000). A multivariable mixed-effects logistic regression were conducted to identify risk factors for EHD and BT. The final model revealed that risk factors associated with EHD and BT seroprevalence may include only age where an age-related increase in seroprevalence was noted for both diseases. Local animal husbandry practices and herd management were not found to be associated with the dynamics of the two diseases. The findings of this study highlight the geographical extent of the disease in the northwestern region and its associated risk factors. It is therefore imperative that further investigations be conducted on vectors and their abudance in order to gain a comprehensive understanding of the current situation.\u003c/p\u003e","manuscriptTitle":"Seroprevalence and risk factors: A comprehensive seroepidemiological study of epizootic hemorrhagic disease and bluetongue in Northwestern Tunisia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 18:50:47","doi":"10.21203/rs.3.rs-7398755/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-07T06:41:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T20:40:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T21:05:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214083905101105321108245672831858182395","date":"2025-09-10T13:15:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262092141823668863561184016147734329043","date":"2025-09-09T22:52:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214296646132776002358972035953762094945","date":"2025-09-09T17:49:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-09T17:27:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-09T07:07:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T06:58:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-05T21:01:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-09-05T20:58:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dde07384-81d1-427e-bed6-858a8187224a","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:02:37+00:00","versionOfRecord":{"articleIdentity":"rs-7398755","link":"https://doi.org/10.1186/s12917-025-05160-6","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2026-01-24 15:59:14","publishedOnDateReadable":"January 24th, 2026"},"versionCreatedAt":"2025-09-17 18:50:47","video":"","vorDoi":"10.1186/s12917-025-05160-6","vorDoiUrl":"https://doi.org/10.1186/s12917-025-05160-6","workflowStages":[]},"version":"v1","identity":"rs-7398755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7398755","identity":"rs-7398755","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}