African swine fever (ASF) is a highly lethal disease of pigs caused by the ASF virus (ASFV), which presents a serious threat to global food security. The movement of contaminated pork products has previously been postulated as contributing to the introduction of ASF into new areas. To evaluate the performance of ASFV detection systems in multi-component pork products, we spiked sausage meat with four different ASFV-containing materials (ASFV cell culture, pork loin, meat juice and bone marrow). DNA was extracted using two manual systems (MagMAX CORE, Qiagen) and one automated (MagMAX CORE) one, and three qPCR assays (VetMAX, King, UPL) were used. The performance of the DNA extraction systems was as follows; automated MagMAX > manual MagMAX > manual Qiagen. The commercial VetMAX qPCR assay yielded significantly lower C_T values (p < 0.001), showing greater sensitivity than the World Organization for Animal Health (OIE)-prescribed assays (King, UPL). Detection probability was the highest for matrices contaminated with bone marrow compared with pork loin or meat juice. An estimated minimum sample size of one 1-g sample is sufficient to detect ASFV in a homogenous pork product if bone marrow from infected pigs comprises 1 part in 10,000. We demonstrated that existing ASFV detection systems are appropriate for use in a food-testing capacity, which can provide an additional control measure for ASF.

Bluetongue is an economically important disease of ruminants caused by the bluetongue virus (BTV). BTV is serologically diverse, which complicates vaccination strategies. Rapid identification of the causative BTV serotypes is critical, however, real-time PCR (RT-qPCR) can be costly and time consuming to perform when the circulating serotypes are unknown. The Luminex xMAP technology is a high-throughput platform that uses fluorescent beads to detect multiple targets simultaneously. We utilized existing BTV serotyping RT-qPCR assays for BTV-1 to BTV-24 and adapted them for use with the xMAP platform. The xMAP assay specifically detected all 24 BTV serotypes when testing reference strains. In all BTV-positive samples, the sensitivity of the BTV xMAP was 87.55% whereas the sensitivity of the serotype-specific RT-qPCR was 79.85%. The BTV xMAP assay allowed for the specific detection of BTV serotypes 1–24 at a lower cost than current RT-qPCR assays. Overall, the assay provides a useful novel diagnostic tool, particularly when analyzing large sample sets. The use of the BTV xMAP assay will allow for the rapid assessment of BTV epidemiology and may inform decision-making related to control and prevention measures.

Kosovo has previously seen two bluetongue (BT) epizootics, each caused by a different serotype, BTV-9 in 2001 and BTV-4 in 2014. Since 2014, no clinical cases of BT have been reported in Kosovo. In September, 2020, clinical signs suggestive of BTV infection were observed in several sheep farms in Kosovo. Blood samples from sheep (n=40) were collected and subjected to further molecular investigations. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in thirty-six sheep from five different farms across two different regions. Full genome sequence analyses indicated that the BTV-4 strains (KOS2020/01 and KOS2020/02) detected in Kosovo in 2020 had high sequence identity (99.9-100) with a strain responsible for an outbreak in North Macedonia in July, 2020, (MKD2020/06) and with previous isolates (99.3) from Greece, Hungary, and France. The percent nucleotide sequence (nt) identity and phylogenetic analyses suggest that the incursion of BTV-4 into Kosovo was a re-emergence of a previously seen strain and not a novel reassortant. This could be due to a reintroduction of the strain into the region or from subclinical circulation which had been ongoing and underreported for years. Surveillance across Kosovo and the Balkan region to monitor the circulation of BTV is crucial if outbreaks are to be brought under control.

Summary: The outbreak of bluetongue virus (BTV) serotype 8 (BTV-8) during 2006-2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV-8 strain re-emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV-8 strain alongside the re-emerging BTV-8 strain isolated from France in 2017. Two groups of eight BTV-naïve British mule sheep were inoculated with 5.75 log ₁₀ TCID ₅₀ ml ⁻¹ of either BTV-8 strain. BTV RNA was detected by 2 dpi in both groups with peak viremia occurring between 5-9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0-8.8 log ₁₀ genome copies mL ⁻¹ ) than the re-emerging BTV-8 strain (2.9-7.9 log ₁₀ genome copies mL ⁻¹ ). All infected sheep developed BTV-specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV-8-inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV-8. In Culicoides sonorensis feeding on the sheep over the period 7-12 dpi, vector competence was significantly higher for the 2007 strain than the re-emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs 1/8) and the overall clinical scores (median 5.25 vs 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re-emerging strain. However, one sheep infected with the re-emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re-emerging BTV-8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry. Summary: The re-emerging Bluetongue virus serotype 8 still presents a threat to naïve ruminants in Europe despite reduced virulence

Outbreaks of bluetongue (BT) virus (BTV) serotype 3 (BTV‐3) have been reported across Europe since late 2023, causing significant levels of disease in sheep, goats and cattle. The infection dynamics and clinical disease severity in the UK cattle and the infection rate of European Culicoides vectors to this BTV‐3 strain have yet to be investigated. Four Holstein‐Friesian calves were infected with BTV‐3 UKG2023/04 using a host:virus:vector transmission model with Culicoides sonorensis biting midges. Clinical signs, gross pathology, infection kinetics, duration of infectious viraemia in the blood and host immune response were assessed. The potential for onwards BTV transmission from infected calves to colony‐reared and the UK field‐caught Culicoides biting midge vectors was quantified over the study period. All four calves were infected with BTV‐3, even when few infected Culicoides had acquired a blood meal. Infected calves experienced mild clinical signs and transient elevation in rectal temperatures, presenting lower disease severity than previously reported in European cattle in the field. BTV infection, measured through the detection of viral RNA by real‐time RT‐PCR of the blood, peaked at 6–10 days post‐infection (dpi) for all individuals. Infectious BTV‐3 was isolated from the blood of three infected calves up to 56 dpi, although the consistency with which BTV was isolated declined after 24 dpi. Protective neutralising antibodies against BTV VP2 were first detected in calves from between 10 and 16 dpi. BTV RNA was detected in colony‐reared and the UK field‐caught Culicoides that had fed on infected calves at peak viraemia (6–8 dpi) and later in infection (42 and 55 dpi). Estimates of the vector competence of the Culicoides obsoletus group for BTV‐3 were low (0.68%). Our data indicate that whilst cattle are less likely than sheep to exhibit clinical disease following infection with BTV‐3, the duration of infectious viraemia observed may permit transmission to Culicoides vectors for several weeks post‐infection.