Molecular Evidence of Epizootic Haemorrhagic Disease Virus and Bluetongue Virus Circulation in Wild Ruminants in Namibia.
DOI:
https://doi.org/10.12834/VetIt.3946.39700.1Keywords:
BTV, EHDV, RT-PCR, Namibia, WildlifeAbstract
Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-borne orbiviruses affecting domestic and wild ruminants. Information on their circulation in Namibian wildlife is limited. This study investigated the molecular detection and serotype distribution of BTV and EHDV in wild ruminants from a commercial game farm in the Khomas Region, Namibia, where wildlife and livestock coexist.
Between June and September 2019, spleen samples from 62 clinically healthy animals (kudu, oryx, and red hartebeest) were analysed by real-time RT-PCR using pan-BTV and pan-EHDV assays, followed by serotype-specific tests for selected BTV types. Two animals (3.23%) tested positive for EHDV. BTV RNA was detected in 24/62 animals (38.71%), with Ct values ranging from 28.3 to 38.4. BTV-3 and BTV-4 were the most frequently identified serotypes, while one sample was positive for BTV-1; six BTV-positive samples remained untyped. High Ct values and low RNA loads likely limited sequencing success.
Although restricted to a single farm and a limited serotype panel, this study provides preliminary molecular evidence of BTV and EHDV circulation in Namibian wild ruminants, highlighting the need for broader epidemiological investigations at the wildlife–livestock interface.
Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are vector-borne viruses transmitted by biting midges of the genus Culicoides (Verwoerd and Erasmus, 2004). Both infect domestic and wild ruminants and belong to the family Sedoreoviridae, genus Orbivirus (Mellor et al., 2009; Matthijnssens et al., 2022). To date, 36 BTV serotypes have been described, including 24 classical and 12 atypical serotypes (Ries et al., 2020; 2021), whereas seven serotypes have been identified for EHDV (Martinez et al., 2025). Both viruses are endemic in many African regions and have repeatedly been introduced into Europe (Cappai et al., 2019; Lorusso et al., 2023; Gondard et al., 2024; Martinez et al., 2025; Plebani et al., 2025; Marcacci et al., 2026). In southern Africa, particularly in the Republic of South Africa, high BTV serotype diversity has been documented (Gerdes, 2004; Coetzee et al., 2012; Van Schalkwyk et al., 2023). EHDV has also been reported in several African countries, including Sudan, South Africa, Kenya, and Zimbabwe (Mohammed and Mellor, 1990; Barnard and Meiswinkel, 1998; Toye et al., 2013; Gordon et al., 2017; Chiuya et al., 2024). In Namibia, information on EHDV circulation is lacking, and molecular data on circulating BTV serotypes are limited. A previous study based on serology identified antibodies against several BTV serotypes in cattle from the Otjozondjupa Region (Molini et al., 2018). However, molecular investigations in wild ruminants are scarce, and no studies have applied real-time RT-PCR directly to field spleen samples from game species.
The aim of this study was to investigate the molecular detection and serotype distribution of BTV and EHDV in wild ruminants from a commercial game farm in the Khomas Region of Namibia, in order to provide preliminary data on virus circulation in a setting where wild and domestic ruminants coexist.
Between June and September 2019, spleen samples were collected from 62 wild ruminants on a commercial livestock and game farm located approximately 45 km from Windhoek. The farm covers about 10,000 hectares and hosts cattle, goats, sheep, and free-ranging game species hunted for meat production. The sampled species were kudu (Tragelaphusstrepsiceros, n = 2), oryx (Oryx gazella, n = 26), and red hartebeest (Alcelaphusbuselaphuscaama, n = 34). All animals were born and raised on the same farm, were older than three years, and appeared clinically healthy at slaughter. This study was conducted on a single farm and included a limited number of animals; therefore, the results reflect only this specific population and cannot be generalised to other regions or management systems. Total RNA was extracted from spleen homogenates using the MagMAX™ CORE Nucleic Acid Purification Kit (Applied Biosystems, Austin, TX, USA) on a KingFisher™ Flex Purification System (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. All samples were screened using the VetMAX™ EHDV Kit (Applied Biosystems, Austin, TX, USA) and the VetMAX™ BTV NS3 All Genotypes Kit (Applied Biosystems, Austin, TX, USA) (pan-EHDV and pan-BTV assays). BTV-positive samples were further tested using (i) the VetMAX™ European BTV Typing Kit (Applied Biosystems, Austin, TX, USA) (serotypes -1, -2, -4, -6, -8, -9, -11, and -16); (ii) an in-house real-time RT-PCR targeting segment 2 of BTV-3 (Lorusso et al., 2018), and (iii) published assays for BTV-25, -26, and -27 (Hoffmann et al., 2010; Maan et al., 2012; Zientara et al., 2014). At the time of the study (2019), validated molecular assays were available only for a subset of classical serotypes. Consequently, not all known BTV serotypes could be investigated. Whole-genome sequencing was attempted on selected samples but was unsuccessful, most likely due to low viral RNA loads. Detection frequencies and 95% confidence intervals were calculated using the binomial Wilson score method.
Two samples tested positive for pan-EHDV (3.23%): one kudu (Ct 38) and one red hartebeest (Ct 37). Twenty-four samples (38.71%) tested positive for pan-BTV, with Ct values ranging from 28.3 to 38.4. Only one sample was positive for BTV-1 (Ct 30). Samples positive for BTV-3 showed Ct values between 25.8 and 38.1, and those positive for BTV-4 between 32.0 and 38.0. Six pan-BTV-positive samples could not be assigned to any of the tested serotypes. BTV-3 and BTV-4 were the most frequently detected serotypes in this population. The distribution of positive results is summarised in Table I. Prevalence estimates are reported in Table II.
| Positive to | Typing | ||||||
| Animal | N° samples (F/M) | EHDV (F/M) | BTV (F/M) | BTV-1 (F/M) | BTV-3 (F/M) | BTV-4 (F/M) | Neg to typing (F/M) |
| Kudu | 2 (2/0) | 1 (1/0) | 1 (1/0) | 0 (0/0) | 0 (0/0) | 1 (1/0) | 0 (0/0) |
| Oryx | 26 (12/14) | 0 (0/0) | 8 (5/3) | 0 (0/0) | 3 (2/1) | 3 (2/1) | 2 (1/1) |
| Red hartebeest | 34 (18/16) | 1 (1/0) | 15 (10/5) | 1 (0/1) | 4 (3/1) | 6 (4/2) | 4 (3/1) |
| TOTAL | 62 ( 32/30) | 2 ( 2/0) | 24 ( 16/8) | 1 ( 0/1) | 7 ( 5/2) | 10 (7/3) | 6 ( 4/2) |
| Animal | EHDV % (95% CI) | BTV % ( 95% CI) | BTV-1 % ( 95% CI) | BTV-3 % ( 95% CI) | BTV-4 % ( 95% CI) | Neg to typing % (95% CI) |
| Kudu | 50.00 (9.59-90.41) | 50.00 (9.59-90.41) | 0.00 (0.00-97.50) | 0.00 (0.00-97.50) | 100.00 (2.50-100.00) | 0.00 (0.00-97.50) |
| Oryx | 0.00 (0.00-12.97) | 30.77 (15.88-50.09) | 0.00 (0.00-36.87) | 37.50 (15.18-65.04) | 37.50 (15.18-65.04) | 25.00 (7.15-59.07) |
| Red hartebeest | 2.94 (0.15-14.58) | 44.12 (28.48-60.53) | 6.67 (1.18-27.23) | 26.67 (11.84-49.22) | 40.00 (21.90-61.27) | 26.67 (11.84-49.22) |
| TOTAL | 3.23 ( 0.89-11.09) | 38.71 ( 26.63-51.05) | 4.17 ( 0.74-20.43) | 29.17 ( 15.65-47.30) | 41.67 ( 25.52-59.98) | 25.00 ( 12.44-44.99) |
Among females, 50.00% (95% CI: 33.63-66.37) tested positive for BTV and 6.25% (95% CI: 1.73-20.15) for EHDV, whereas among males 26.67% (95% CI: 14.18-44.94) tested positive for BTV and none for EHDV. These figures should be interpreted descriptively, given the limited sample size and the study design.
The relatively high Ct values observed in several samples (up to 38.4) indicate low amounts of viral RNA. Such results should be interpreted cautiously, as high Ct values may reflect low-level viral RNA detection, residual viral material, or subclinical infection, rather than active viral replication. The low RNA concentrations likely contributed to the unsuccessful sequencing attempts and prevented further characterisation of untyped strains.
Compared with previous serological evidence from Namibia (Molini et al., 2018), the present study provides molecular confirmation of BTV circulation in wild ruminants on a game farm. However, given the single-farm design, the limited number of animals, and the restricted panel of molecular assays, no conclusions can be drawn regarding regional prevalence, temporal trends, or the full spectrum of circulating serotypes. The identity of untyped strains remains undetermined, and further investigations using broader molecular panels and higher-quality samples would be required to clarify their classification.
A higher proportion of females tested positive for BTV compared to males in this dataset. However, the study was not designed to assess sex-related risk factors, and the sample size does not allow robust statistical inference. Therefore, this observation should be considered descriptive only.
In conclusion, this study provides preliminary molecular evidence of BTV and EHDV RNA detection in wild ruminants from a commercial game farm in central Namibia. The findings suggest BTV circulation in this specific setting, while EHDV detection was limited. Broader studies including multiple farms, larger sample sizes, and comprehensive molecular characterisation are needed to better understand the epidemiology of these orbiviruses in Namibian wildlife and their potential interface with domestic livestock.
Ethical approval
No ethical approval was required. All included samples were collected from carcasses in an abattoir.
Conflict of interest
The authors do not have any conflict of interest.
Author Contributions
Conceptualization: Umberto Molini; Methodology: Umberto Molini, Alessio Lorusso, and Ottavio Portanti; Formal analysis: Umberto Molini, Maria Yvonne Hemberger, Nicandro Rodi, Mariassunta Iannetta, and Ottavio Portanti; Investigation: Maria Yvonne Hemberger, and Juliet Kabajani; Writing original draft preparation: Umberto Molini, and Gloria Plebani; Writing, review and editing: Umberto Molini, Gloria Plebani, Alessio Lorusso, and Ottavio Portanti; Supervision: Umberto Molini
All authors have read and agreed to the published version of the manuscript.
Fundings
This work was partially funded by the Italian Ministry of Health through the project “CARBO – Caratterizzazione biologica e fattori di virulenza di nuovi e vecchi arbovirus animali”, grant code MSRCTE0223.
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