Bluetongue is an infectious, non-contagious, arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV), producing severe impacts on livestock. Historically, Southern Europe has suffered multiple incursions of different BTV serotypes with serious consequences. In 2021, BTV re-emerged in the Balearic Islands (Spain) after 16 years free of the disease, causing a large outbreak that mainly affected sheep, as well as cattle and goats. A novel emerging strain of BTV serotype 4 (BTV-4) was identified via preliminary molecular characterization as the etiological culprit of the epizootic. This study delineates the outbreak in the Balearic Islands in 2021, encompassing field-based clinical observations and laboratory findings. Additionally, an experimental infection was conducted in sheep using the novel BTV-4 strain to assess its virulence, pathogenicity, and laboratory diagnostic characteristics. The in vivo characterization was conducted concurrently with the virulent and widely disseminated BTV-4 RNM 2020 strain that has precipitated significant outbreaks in the Mediterranean region in recent years. Both strains exhibited analogous pathogenic potential in sheep and yielded equivalent outcomes in diagnostic parameters. Furthermore, the impact of the novel BTV-4 strain is discussed.

In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays ‘polyvalent live attenuated’ is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015–2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987–1990), were included in the study for the validation of these methods.

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease of ruminants, and has a severe impact on livestock. It is caused by Bluetongue virus (BTV), a double-stranded (ds) RNA virus with a segmented genome (10 segments), belonging to the Seoreoviridae family, Orbivirus genus. Over the last 25 years, Europe has suffered multiple incursions of different BTV serotypes with serious consequences, which have mainly been controlled thanks to vaccination. In the case of Spain, from 2000 to 2023, BTV serotypes 1, 2, 4 and 8 have caused epidemics, and, sporadically, BTV-1 and -4 were detected in the same area and period. In 2024, BTV serotypes 1, 3 and 8 circulated simultaneously in the southwest of the country, causing a severe clinical impact in sheep but also in cattle and a multitude of outbreaks. Additionally, despite vaccination, serotype 4 also circulated that year, especially in areas where the other serotypes were already circulating. Whole-genome sequencing and phylogenetic analyses allowed us to confirm that serotypes 1 and 4 were homologous to viruses circulating in the country since 2000s, while serotypes 3 and 8 were homologous to BTVs recently notified in neighboring countries. In this context, many BTV co-infections of two or more different serotypes were confirmed by serotype-specific RT-PCRs, both in farms and individual animals. An epidemic caused by four serotypes coinciding in space and time had never occurred before in Spain, being a challenge for the diagnosis and control of this disease. Moreover, it could have favored the appearance of reassortant viruses with an unknown virulence, posing an additional risk. The data presented here raise the question of whether the co-circulation of different BTV strains, an exceptional situation, could become the new normal in certain areas of Europe.

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organisation for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Agüero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH Reference Laboratory for AHS in the Republic of South Africa (RSA) reported an AHSV variant undetected by the Agüero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Agüero method was validated by the WOAH Reference Laboratories in Spain and the UK, employing a broad collection of AHSV strains and clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Agüero versus the WOAH reference rRT-PCRs showed that the modified method exhibited good diagnostic performance and enabled detection of the new RSA AHSV variant nucleic acid.