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.

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.

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.

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.

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.

Complete genome sequences of epizootic hemorrhagic disease virus serotypes 5 and 6 isolated in Japan.

Here, we report the complete genome sequences of epizootic hemorrhagic disease (EHD) virus serotypes 5 (EHDV-5) and 6 (EHDV-6) isolated in the Yaeyama Islands of Okinawa Prefecture, Japan. The EHDV-5 strain, ON-11/E/16, which was isolated in 2016, is, to our knowledge, the second EHDV-5 strain to be isolated after the first was isolated in Australia in 1977. In each of the genome segments, ON-11/E/16 was most closely related to EHDV strains of different serotypes isolated in Australia and Japan. Our results support the idea that various serotypes of EHDV have been circulating while causing reassortment in the Asia-Pacific region. In all genome segments, the EHDV-6 strain, ON-3/E/14, which was isolated in 2014, was highly similar to EHDV-6 strain HG-1/E/15, which was detected in affected cattle during the EHD epidemic in Hyogo prefecture in 2015. Therefore, these two EHDV-6 strains, ON-3/E/14 and HG-1/E/15, may have the same origin. However, it is unclear whether EHDV-6 was transmitted directly between the locations where those strains were isolated/detected (approx. 1,500 km apart) or whether EHDV-6 strains of the same origin entered each location at different times. In addition, we cannot rule out the possibility that EHDV-6 infection has spread unnoticed through asymptomatic cattle in other areas of Japan. Therefore, further investigation into EHDV infection in cattle is necessary for a more detailed understanding of the ecology of EHDV in Japan.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

Circulation of Bluetongue Virus Serotypes 1, 4, 8, 10 and 16 and Epizootic Hemorrhagic Disease Virus in the Sultanate of Oman in 2020-2021.

The circulation of Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) in the Middle East has already been reported following serological analyses carried out since the 1980s, mostly on wild ruminants. Thus, an EHD virus (EHDV) strain was isolated in Bahrain in 1983 (serotype 6), and more recently, BT virus (BTV) serotypes 1, 4, 8 and 16 have been isolated in Oman. To our knowledge, no genomic sequence of these different BTV strains have been published. These same BTV or EHDV serotypes have circulated and, for some of them, are still circulating in the Mediterranean basin and/or in Europe. In this study, we used samples from domestic ruminant herds collected in Oman in 2020 and 2021 for suspected foot-and-mouth disease (FMD) to investigate the presence of BTV and EHDV in these herds. Sera and whole blood from goats, sheep and cattle were tested for the presence of viral genomes (by PCR) and antibodies (by ELISA). We were able to confirm the presence of 5 BTV serotypes (1, 4, 8, 10 and 16) and the circulation of EHDV in this territory in 2020 and 2021. The isolation of a BTV-8 strain allowed us to sequence its entire genome and to compare it with another BTV-8 strain isolated in Mayotte and with homologous BTV sequences available on GenBank.

Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013-2017, China.

Epizootic hemorrhagic disease (EHD) is an infectious viral disease caused by epizootic hemorrhagic disease virus (EHDV) and EHDV frequently circulates in wild and domestic ruminants. Sporadic outbreaks of EHD have caused thousands of deaths and stillbirths on cattle farms. However, not much is known about the circulating status of EHDV in Guangdong, southern China. To estimate the seroprevalence of EHDV in Guangdong province, 2886 cattle serum samples were collected from 2013 to 2017 and tested for antibodies against EHDV using a competitive ELISA. The overall seroprevalence of EHDV reached 57.87% and was highest in autumn (75.34%). A subset of positive samples were serotyped by a serum neutralization test, showing that EHDV serotypes 1 and 5-8 were circulating in Guangdong. In addition, EHDV prevalence always peaked in autumn, while eastern Guangdong had the highest EHDV seropositivity over the five-year period, displaying apparent temporal-spatial distribution of EHDV prevalence. A binary logistic model analysis indicated a significant association between cattle with BTV infections and seroprevalence of EHDV (OR = 1.70, p < 0.001). The co-infection of different serotypes of EHDV and BTV raises a high risk of potential genomic reassortment and is likely to pose a significant threat to cattle, thus urging more surveillance to monitor their circulating dynamics in China.

Abundance and diversity of Culicoides Latreille (Diptera: Ceratopogonidae) in southern Ontario, Canada.

BACKGROUND: Culicoides Latreille (Diptera: Ceratopogonidae) is a genus of hematophagous midges feeding on various vertebrate hosts and serving as a vector for numerous pathogens important to livestock and wildlife health. North American pathogens include bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses. Little is known about Culicoides spp. distribution and abundance and species composition in Ontario, Canada, despite bordering numerous U.S. states with documented Culicoides spp. and BT and EHD virus activity. We sought to characterize Culicoides spp. distribution and abundance and to investigate whether select meteorological and ecological risk factors influenced the abundance of Culicoides biguttatus, C. stellifer, and the subgenus Avaritia trapped throughout southern Ontario. METHODS: From June to October of 2017 to 2018, CDC-type LED light suction traps were placed on twelve livestock-associated sites across southern Ontario. Culicoides spp. collected were morphologically identified to the species level when possible. Associations were examined using negative binomial regression among C. biguttatus, C. stellifer, and subgenus Avaritia abundance, and select factors: ambient temperature, rainfall, primary livestock species, latitude, and habitat type. RESULTS: In total, 33,905 Culicoides spp. midges were collected, encompassing 14 species from seven subgenera and one species group. Culicoides sonorensis was collected from three sites during both years. Within Ontario, the northern trapping locations had a pattern of seasonal peak abundance in August (2017) and July (2018), and the southern locations had abundance peaks in June for both years. Culicoides biguttatus, C. stellifer, and subgenus Avaritia were significantly more abundant if ovine was the primary livestock species at trapping sites (compared to bovine). Culicoides stellifer and subgenus Avaritia were significantly more abundant at mid- to high-temperature ranges on trap days (i.e., 17.3-20.2 and 20.3-31.0 °C compared to 9.5-17.2 °C). Additionally, subgenus Avaritia were significantly more abundant if rainfall 4 weeks prior was between 2.7 and 20.1 mm compared to 0.0 mm and if rainfall 8 weeks prior was between 0.1 and 2.1 mm compared to 0.0 mm. CONCLUSIONS: Results from our study describe Culicoides spp. distribution in southern Ontario, the potential for spread and maintenance of EHD and BT viruses, and concurrent health risks to livestock and wildlife in southern Ontario in reference to certain meteorological and ecological risk factors. We identified that Culicoides spp. are diverse in this province, and appear to be distinctly distributed spatially and temporally. The livestock species present, temperature, and rainfall appear to have an impact on the abundance of C. biguttatus, C. stellifer, and subgenus Avaritia trapped. These findings could help inform targeted surveillance, control measures, and the development of management guides for Culicoides spp. and EHD and BT viruses in southern Ontario, Canada.

Host associations of biting midges (Diptera: Ceratopogonidae: Culicoides) at deer farms in Florida, USA.

Documenting the host use of vector species is important for understanding the transmission dynamics of vector-borne pathogens. Biting midges (Diptera: Ceratopogonidae: Culicoides) are vectors of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) worldwide. However, relative to mosquitoes and many other vector groups, host associations of this group are poorly documented. In this study, we used PCR-based bloodmeal analysis to determine species-level host associations of 3,603 blood-engorged specimens belonging to 18 Culicoides species at 8 deer farms in Florida, USA. We used a binomial mixed model with a Bayesian framework to compare the effect of host composition on the feeding patterns of Culicoides spp. and employed the Morisita-Horn Index to investigate the similarity of host use between farms for Culicoides stellifer and Culicoides insignis. Results show that the estimated probability of Culicoides spp. feeding upon white-tailed deer depends on the availability of cattle or exotic game and demonstrates differences in host-feeding selection among species. Culicoides insignis had high host similarity across farms suggesting that its host-use patterns are somewhat conserved. Culicoides stellifer had lower host similarity across farms suggesting that it is a more opportunistic feeder. White-tailed deer are fed upon by many Culicoides species on deer farms in Florida, and while most Culicoides species feed on white-tailed deer, the ratio of white-tailed deer bloodmeals to other bloodmeals is likely influenced by host availability. Culicoides spp. taking a majority of their bloodmeals from farmed white-tailed deer should be assessed for their vector competence for EHDV and BTV.

Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

West Nile Virus and Epizootic Hemorrhagic Disease Virus Co-Infection in a Novel Host at the Nashville Zoo.

On August 30, 2017, one of five bontebok in a mixed-species exhibit at the Nashville Zoo at Grassmere exhibited acute hind-limb ataxia and altered demeanor. Pathological examination demonstrated meningoencephalitis and spinal myelitis. Coinfection of West Nile virus (WNV) and epizootic hemorrhagic disease virus (EHDV) was revealed by quantitative real-time and traditional reverse transcription-polymerase chain reaction assays and virus isolation/whole genome sequencing from brain tissue, respectively. Whole genome sequencing was conducted for EHDV. Mosquito testing from September 19 to October 13, 2017, demonstrated a higher WNV infection rate in mosquitoes at the zoo compared with the rest of Nashville-Davidson County. EHDV is endemic in wild white-tailed deer (family Cervidae) in Tennessee, and the prevalence in wildlife depends on environmental influences. This case illustrates the potential susceptibility of exotic zoo animals to endemic domestic arthropod-borne viruses (arboviruses) and reinforces the importance of cooperative antemortem and postmortem surveillance strategies among human, wildlife, and domestic animal health agencies.

The Impact of Variation in the Toll-like Receptor 3 Gene on Epizootic Hemorrhagic Disease in Illinois Wild White-Tailed Deer (Odocoileus virginianus).

Epizootic hemorrhagic disease (EHD) leads to high mortality in white-tailed deer (Odocoileus virginianus) and is caused by a double-stranded RNA (dsRNA) virus. Toll-like receptor 3 (TLR3) plays a role in host immune detection and response to dsRNA viruses. We, therefore, examined the role of genetic variation within the TLR3 gene in EHD among 84 Illinois wild white-tailed deer (26 EHD-positive deer and 58 EHD-negative controls). The entire coding region of the TLR3 gene was sequenced: 2715 base pairs encoding 904 amino acids. We identified 85 haplotypes with 77 single nucleotide polymorphisms (SNPs), of which 45 were synonymous mutations and 32 were non-synonymous. Two non-synonymous SNPs differed significantly in frequency between EHD-positive and EHD-negative deer. In the EHD-positive deer, phenylalanine was relatively less likely to be encoded at codon positions 59 and 116, whereas leucine and serine (respectively) were detected less frequently in EHD-negative deer. Both amino acid substitutions were predicted to impact protein structure or function. Understanding associations between TLR3 polymorphisms and EHD provides insights into the role of host genetics in outbreaks of EHD in deer, which may allow wildlife agencies to better understand the severity of outbreaks.

The Effects of Light Wavelength and Trapping Habitat on Surveillance of Culicoides Biting Midges (Diptera: Ceratopogonidae) in Alabama.

In the southeastern United States, biting midges transmit agents of hemorrhagic diseases that are enzootic among white-tailed deer (Odocoileus virginianus (Zimmermann), Artiodactyla: Cervidae). Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), the only confirmed vector of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) in the United States, is rarely collected in the Southeast, implying that other Culicoides Latreille species act as vectors. Despite multiple surveillance studies, the influence of trapping habitat and light wavelength on Culicoides sampling has yet to be investigated in Alabama. This study sampled Culicoides species at a deer research facility using CO2-baited CDC light traps with three distinct wavelengths. Traps were rotated within three habitats to examine impacts of habitat type and light wavelength on Culicoides abundance and parity status. For most species, midges were more abundant in a pine forest compared to a hardwood-forest riparian zone or a lightly wooded area adjacent to a seasonal pond. The pine forest generally had negative effects on parity status, suggesting that most females in this habitat were foraging for their first bloodmeal. Ultraviolet (UV) black-light (350 nm-360 nm) attracted more midges than incandescent light or UV LED light (385 nm-395 nm), but wavelength had less of an effect on parity than habitat. This study indicates that light wavelength and habitat significantly influence Culicoides sampling outcomes, and that when collecting parous females is desired (e.g., EHDV/BTV surveillance), targeting areas around oviposition sites may be a better strategy than trapping where midges are most abundant.

Production and Characterization of Monoclonal Antibodies Against the VP7 Protein of Epizootic Hemorrhagic Disease Virus.

Monoclonal antibodies (MAbs) against epizootic hemorrhagic disease virus (EHDV) were produced by immunizing BALB/c mice with rec-VP7-EHDV2; 66 clones producing MAbs able to recognize the VP7-EHDV with a strong reaction were obtained and tested in indirect enzyme-linked immunosorbent assay (i-ELISA) against the whole epizootic hemorrhagic disease (EHD) virus serotype 2; potential cross-reactions with related orbiviruses, as Bluetongue virus (BTV) and African horse sickness virus (AHSV), were investigated as well by i-ELISA, Western blot, and immunofluorescence. Fifty-three MAbs were specific for EHDV (VP7 recombinant protein and whole virus) and 13 reacted also with the VP7 of BTV. None of the MAbs reacted with AHSV. MAbs specific for EHDV were further characterized in a competitive ELISA (c-ELISA): 20 among them were found useful to develop a c-ELISA for the detection of antibodies against EHDV in bovine sera. The availability of this extensive set of MAbs provides the opportunity to develop a c-ELISA for the serological diagnosis of EHDV and to tune new methods for the isolation and identification of the virus in biological samples and cell cultures. The experimentation protocol was approved by the Italian Ministry of Health (number 639/2018-PR, Resp. to Prot. BDF16.13#295833199#).

Morphological and Molecular Identification of Culicoides (Diptera: Ceratopogonidae) Species of the Southern California Desert.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are vectors of important animal pathogens including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). While some Culicoides species present in the southern California desert are implicated in the transmission of these viruses to ruminant animals, these species have not been extensively studied due in part to the challenge of identifying Culicoides to species and to the lack of published gene sequences for these species to support their molecular identification. In this study, Culicoides were captured using suction traps baited with either carbon dioxide or UV light from transitional habitat between the southern California peninsular mountain ranges and the Colorado desert of southeastern California. Captured midges were initially identified using traditional morphological methods, with species identification subsequently confirmed by sequence analysis of COI and 28S rDNA genes. Phylogenetic analyses support that some Culicoides subgenera are not monophyletic. Two recognized species (C. sitiens Wirth and Hubert and C. bakeri Vargas) shared the same COI and 28S sequences. An additional cryptic species may be present within C. sitiens. Two additional recognized species (C. cacticola Wirth and Hubert and C. torridus Wirth and Hubert) may be conspecific or cryptic to each other. In total, 19 Culicoides species (or species aggregate) were collected in this study, with genetic sequences published for the first time for 16 of them. Published genetic sequences will support future research on these species, including studies on the ecology and habits of their immature stages which are often tedious to identify using morphology.