Epizootic Hemorrhagic Disease Virus Research Articles

Culicoides Midge Abundance across Years: Modeling Inter-Annual Variation for an Avian Feeder and a Candidate Vector of Hemorrhagic Diseases in Farmed Wildlife

(1) Background: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are orbiviruses that cause hemorrhagic disease (HD) with significant economic and population health impacts on domestic livestock and wildlife. In the United States, white-tailed deer (Odocoileus virginianus) are particularly susceptible to these viruses and are a frequent blood meal host for various species of Culicoides biting midges (Diptera: Ceratopogonidae) that transmit orbiviruses. The species of Culicoides that transmit EHDV and BTV vary between regions, and larval habitats can differ widely between vector species. Understanding how midges are distributed across landscapes can inform HD virus transmission risk on a local scale, allowing for improved animal management plans to avoid suspected high-risk areas or target these areas for insecticide control. (2) Methods: We used occupancy modeling to estimate the abundance of gravid (egg-laden) and parous (most likely to transmit the virus) females of two putative vector species, C. stellifer and C. venustus, and one species, C. haematopotus, that was not considered a putative vector. We developed a universal model to determine habitat preferences, then mapped a predicted weekly midge abundance during the HD transmission seasons in 2015 (July–October) and 2016 (May–October) in Florida. (3) Results: We found differences in habitat preferences and spatial distribution between the parous and gravid states for C. haematopotus and C. stellifer. Gravid midges preferred areas close to water on the border of well and poorly drained soil. They also preferred mixed bottomland hardwood habitats, whereas parous midges appeared less selective of habitat. (4) Conclusions: If C. stellifer is confirmed as an EHDV vector in this region, the distinct spatial and abundance patterns between species and physiological states suggest that the HD risk is non-random across the study area.

Patterns of Hemorrhagic Disease in White-Tailed Deer (Odocoileus Virginianus) in the Great Plains of the USA, 1982-2020.

Hemorrhagic disease (HD) of deer is caused by epizootic hemorrhagic disease virus (EHDV) or bluetongue virus (BTV) and is considered one of the most important viral diseases of white-tailed deer (Odocoileus virginianus). Despite evidence of changing patterns of HD in the northeastern and upper midwestern US, the historical and current patterns of HD in the Great Plains remain poorly described. We used results from an annual survey documenting HD mortality to characterize historic and current patterns of HD in the northern and central Great Plains (North Dakota, South Dakota, Nebraska, Kansas, and Oklahoma), US, between 1982 and 2020. Further, we assessed temporal change using linear regression to determine change in annual reporting intensity (percentage of counties in a state with reported HD) and change in reporting frequency (the number of years a county or state reported HD) during each decade between 1982 and 2020. Across the 38-yr study period, HD reports expanded northeast across latitude and longitude. Intensity of HD reports significantly increased during this period for three (North Dakota, South Dakota, Kansas) of five states examined. Frequency of reports also increased for all five states. Such changes in northern latitudes might lead to increased deer mortality in regions where HD epizootics have been historically less frequent. Understanding how patterns of HD are changing on the landscape is important when considering future deer management in the face of other mortality factors.

Transcriptomic Investigation of the Virus Spectrum Carried by Midges in Border Areas of Yunnan Province

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (−ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases.

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3–8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021-2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies.

Evidence of Active Orbivirus Transmission in 2016 in Kansas and Nebraska.

Retrospective serological and case diagnostic data of endemic bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) provide evidence of viral transmission among livestock and wildlife from 2016 in Kansas and Nebraska. Serological testing of mature cattle in nine distinct regional zones of Kansas revealed 76% to 100% had detectable antibodies to BTV and/or EHDV. Specimens tested in the Kansas Veterinary Diagnostic Laboratory (55 submissions) were 51% test positive for antibodies to BTV and/or EHDV. Specimens tested in the Nebraska Veterinary Diagnostic Center (283 submissions) were 25% test positive for antibodies to BTV and/or EHDV. Low disease incidence in white-tailed deer and other susceptible wild ungulates was observed during 2016. However, there were no confirmed reports of disease in livestock in either state. The reasons for emergence of significant clinical disease in livestock and wildlife populations remain undefined.

The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023.

Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease seriously affecting cattle and some wild ruminants and has a worldwide distribution. All viruses can be subdivided into "Eastern" and "Western" topotypes according to geographic distribution via the phylogenetic analysis of internal genes. In Israel, during the last decade, three outbreaks were registered: caused by EHDV-6 in 2015, by EHDV-1 in 2016, and by EHDV-7 in 2020. Additionally, RNA of EHDV-8 was found in imported calves from Portugal in 2023. During the same period in other countries of the region, non-Israeli-like EHDV-6 and EHDV-8 were identified. Full genome sequencing, BLAST, and phylogenetic analyses of the locally and globally known EHDV genomes allowed us to presume the probable route and origin of these viruses detected in Israel. Thus, EHDV-6 has probably been circulating in the region for a long period when EHDV-1 and -8 appeared here for the last years, while their route of introduction into the new areas was probably natural; all of them belonged to the "Western" topotype. In contrast, EHDV-7 probably had the "Eastern", anthropogenic origin. Data from the study can facilitate the evaluation of the appearance or reappearance of EHDVs in the Mediterranean area and enhance the planning of prevention measures.

The Study of Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) Circulation and Vectors at the Municipal Parks and Zoobotanical Foundation of Belo Horizonte, Minas Gerais, Brazil (FPMZB-BH).

Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) are Orbiviruses primarily transmitted by their biological vector, Culicoides spp. Latreille, 1809 (Diptera: Ceratopogonidae). These viruses can infect a diverse range of vertebrate hosts, leading to disease outbreaks in domestic and wild ruminants worldwide. This study, conducted at the Belo Horizonte Municipal Parks and Zoobotany Foundation (FPMZB-BH), Minas Gerais, Brazil, focused on Orbivirus and its vectors. Collections of Culicoides spp. were carried out at the FPMZB-BH from 9 December 2021 to 18 November 2022. A higher prevalence of these insects was observed during the summer months, especially in February. Factors such as elevated temperatures, high humidity, fecal accumulation, and proximity to large animals, like camels and elephants, were associated with increased Culicoides capture. Among the identified Culicoides spp. species, Culicoides insignis Lutz, 1913, constituted 75%, and Culicoides pusillus Lutz, 1913, 6% of the collected midges, both described as competent vectors for Orbivirus transmission. Additionally, a previously unreported species in Minas Gerais, Culicoides debilipalpis Lutz, 1913, was identified, also suspected of being a transmitter of these Orbiviruses. The feeding preferences of some Culicoides species were analyzed, revealing that C. insignis feeds on deer, Red deer (Cervus elaphus) and European fallow deer (Dama dama). Different Culicoides spp. were also identified feeding on humans, raising concerns about the potential transmission of arboviruses at the site. In parallel, 72 serum samples from 14 susceptible species, including various Cervids, collected between 2012 and 2022 from the FPMZB-BH serum bank, underwent Agar Gel Immunodiffusion (AGID) testing for BTV and EHDV. The results showed 75% seropositivity for BTV and 19% for EHDV. Post-testing analysis revealed variations in antibody presence against BTV in a tapir and a fallow deer and against EHDV in a gemsbok across different years. These studies confirm the presence of BTV and EHDV vectors, along with potential virus circulation in the zoo. Consequently, implementing control measures is essential to prevent susceptible species from becoming infected and developing clinical diseases.

Seven-year prospective study on yearly incidence of Orbivirus infection of captive white-tailed deer and potential Culicoides vectors.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that are transmitted by biting midges in the genus Culicoides (Diptera: Ceratopogonidae) and can cause hemorrhagic disease in certain ruminants. The objectives of this study were to measure the incidence of BTV and EHDV infections in captive white-tailed deer herd as well as tissues and corresponding presence of Culicoides midges at a location near Clinton, LA. During a 7-yr study with yearly outbreaks of hemorrhagic disease in the deer herd, 15 species of Culicoides were captured using Centers for Disease Control (CDC) black light traps. Reverse transcriptase quantitative polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in pools of midges and tissues of deer. From 2012 to 2018, 1,711 pools of midges representing 24,859 specimens were tested, and specimens from 5 of the 15 collected species (Culicoides debilipalpis, Culicoides stellifer, Culicoides venustus, Culicoides haematopotus, and Culicoides crepuscularis) were found to be PCR positive for BTV and EHDV. Most of the BTV-positive pools of biting midges were from specimens of C. debilipalpis and C. stellifer, and most of the EHDV-positive pools were from specimens of C. venustus and C. stellifer. During the 7-yr period, 112 white-tailed deer that died at the study location were PCR positive for BTV or EHDV: detected BTV serotypes were 10 and 12 and EHDV serotypes were 1, 2, and 6. There was a significant increase in BTV/EHDV antibody prevalence in white-tailed deer during the study; antibody-positive rates increased from 15% to 78% in the deer herd of approximately 100 animals.

Isolation of Epizootic Hemorrhagic Disease Virus Serotype 10 from Culicoides tainanus and Associated Infections in Livestock in Yunnan, China.

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6-99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus's presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health.

Viral diversity and blood-feeding patterns of Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae).

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited. Adult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS). Analysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool. The results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats. Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

From forests to fields: investigating Culicoides (Diptera: Ceratopogonidae) abundance and diversity in cattle pastures and adjacent woodlands.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are hematophagous flies that feed on wild and domestic ruminants. They can transmit arboviruses, such as bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), which circulate in the United States. Larvae occupy a range of aquatic and semiaquatic habitats, and disperse short distances from their development sites. In the southeastern United States, there are limited studies on the abundance and diversity of Culicoides in wooded and adjacent livestock pasture habitats. In this study, we characterized Culicoides diversity and abundance within these distinct habitat types. BG-Sentinel and CDC miniature suction traps baited with CO2 or UV-light were placed in wooded and pasture habitats at 2 locations on a university beef farm in Savoy, Arkansas. Traps were set once per week for 9 wk during August-October of 2021 and 2022. Fifteen species were collected during this study, and the 2 most abundant species were Culicoides haematopotus Malloch and Culicoides stellifer Coquillett. There was a significant effect of site and location on C. haematopotus collections, and a significant effect and interaction of site and trap on C. stellifer collections. In the woods, significantly more C. stellifer were collected from CDC-UV traps, while in the pasture significantly more were collected in CDC-CO2 traps. These data suggest that C. stellifer, a putative vector of BTV and EHDV in the southeast, may be traveling into the pasture to host-seek, while C. haematopotus remains primarily in wooded areas. This study reveals community differences between these habitat types and implications for Culicoides control.

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

Culicoides-borne Orbivirus epidemiology in a changing climate.

Orbiviruses are of significant importance to the health of wildlife and domestic animals worldwide; the major orbiviruses transmitted by multiple biting midge (Culicoides) species include bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus. The viruses, insect vectors, and hosts are anticipated to be impacted by global climate change, altering established Orbivirus epidemiology. Changes in global climate have the potential to alter the vector competence and extrinsic incubation period of certain biting midge species, affect local and long-distance dispersal dynamics, lead to range expansion in the geographic distribution of vector species, and increase transmission period duration (earlier spring onset and later fall transmission). If transmission intensity is associated with weather anomalies such as droughts and wind speeds, there may be changes in the number of outbreaks and periods between outbreaks for some regions. Warmer temperatures and changing climates may impact the viral genome by facilitating reassortment and through the emergence of novel viral mutations. As the climate changes, Orbivirus epidemiology will be inextricably altered as has been seen with recent outbreaks of bluetongue, epizootic hemorrhagic disease, and African horse sickness outside of endemic areas, and requires interdisciplinary teams and approaches to assess and mitigate future outbreak threats.

Wing morphometrics of biting midges (Diptera: Culicoides) of veterinary importance in Madagascar.

Biting midges are vectors of arboviruses such as bluetongue virus, bovine ephemeral fever virus, Akabane virus, African horse sickness virus, epizootic haemorrhagic disease virus and Schmallenberg virus. Fast and accurate identification of biting midges is crucial in the study of Culicoides-borne diseases. Morphological identification of biting midges has revealed the presence of cryptic species. A total of 20 species are reported in Madagascar. In this study, we assessed wing morphometric analysis for identification of seven species namely C. dubitatus Kremer, Rebholtz-Hirtzel and Delécolle, C. enderleini Cornet and Brunhes, C. kibatiensis Goetghebuer, C. miombo Meiswinkel, C. moreli Clastrier, C. nevilli Cornet and Brunhes, and C. zuluensis de Meillon. Culicoides enderleini, C. miombo, C. moreli, C. nevilli and C. zuluensis are vectors diseases. A molecular approach, based on the cytochrome oxidase I gene (Cox1), was used for species delimitation. The molecular analysis presented seven different clades grouped two-by-two according to morphological characters. A total of 179 wing images were digitised. We found morphometric variation among seven species based on 11 landmarks and two outlines. Wing shape variation plots showed that species overlapped with species belonging to the same group. The cross-validation revealed a relatively high percentage of correct classification in most species, ranging from 91.3% to 100% for landmarks; 60% to 82.6% for outlines-1 and 77.1% to 91.3% for outlines-2. Our study suggests that wing geometric morphometric analysis is a robust tool for reliable "Moka Fohy" identification in Madagascar. This inexpensive and simple method is a precise supplement to morphological identification, with reaches the accuracy of Cox1 barcoding.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV‑8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV‑8, sharing the same transmission patterns of BTV, can also spread to Europe.

Development and validation of an RT-qPCR for detection and quantitation of emerging epizootic hemorrhagic disease virus serotype 8 RNA from field samples

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control.

Outbreak of epizootic hemorrhagic disease in captive reindeer (Rangifer tarandus).

In September 2020, an outbreak of epizootic hemorrhagic disease occurred in captive reindeer (Rangifer tarandus) and was associated with neurological signs and mortality. Four reindeer died or were euthanized after acute illness over a 12-day period. Affected reindeer displayed abnormal behavior, neurologic signs, lethargy, and/or lameness. The most consistent gross finding was dark red streaks throughout the adrenal gland cortices (4/4). One animal had acute hemorrhage involving the subcutis and skeletal muscles over the ventrolateral body wall and back, and abomasal serosa. Histologically, the most common lesions were adrenal gland cortical hemorrhage (4/4) with necrosis (3/4) and lymphoplasmacytic meningoencephalitis with gliosis, glial nodules, satellitosis, and nonsuppurative perivascular cuffing (4/4). The brain lesions were most frequent in the gray matter of the cerebrum, hippocampus, and thalamus but also involved the cerebellum and brainstem. Epizootic hemorrhagic disease virus serotype 6 was detected through PCR and sequencing of the spleen in all cases.

A microfluidic biosensor for the diagnosis of chronic wasting disease

Cervids are affected by a neurologic disease that is always fatal to individuals and has population effects. This disease is called chronic wasting disease (CWD) and is caused by a misfolded prion protein. The disease is transmitted via contact with contaminated body fluids and tissue or exposure to the environment, such as drinking water or food. Current CWD diagnosis depends on ELISA screening of cervid lymph nodes and subsequent immunohistochemistry (IHC) confirmation of ELISA-positive results. The disease has proven to be difficult to control in part because of sensitivity and specificity issues with the current test regimen. We have investigated an accurate, rapid, and low-cost microfluidic microelectromechanical system (MEMS) biosensing device for the detection of CWD pathologic prions in retropharyngeal lymph nodes (RLNs), which is the current standard type of CWD diagnostic sample. The device consists of three novel regions for concentrating, trapping, and detecting the prion. The detection region includes an array of electrodes coated with a monoclonal antibody against pathologic prions. The experimental conditions were optimized using an engineered prion control antigen. Testing could be completed in less than 1 hour with high sensitivity and selectivity. The biosensor detected the engineered prion antigen at a 1:24 dilution, while ELISA detected the same antigen at a 1:8 dilution. The relative limit of detection (rLOD) of the biosensor was a 1:1000 dilution of a known strong positive RLN sample, whereas ELISA showed a rLOD of 1:100 dilution. Thus, the biosensor was 10 times more sensitive than ELISA, which is the currently approved CWD diagnostic test. The biosensor’s specificity and selectivity were confirmed using known negative RPLN samples, a negative control antibody (monoclonal antibody against bovine coronavirus BCV), and two negative control antigens (bluetongue virus and Epizootic hemorrhagic disease virus). The biosensor’s ability to detect pathogenic prions was verified by testing proteinase-digested positive RLN samples.