Culicoides Midges Research Articles

Potential mechanisms underlying bluetongue virus emergence and spread

Bluetongue is a non-zoonotic arboviral disease transmitted by biting midges of the genus Culicoides. Among 1,347 Culicoides species, 30 are known to be vectors of the disease. Bluetongue affects domestic and wild ruminants across all continents, except the Antarctica, causing growing economic losses in livestock production and international trade. In this work, we focused on potential mechanisms underlying the bluetongue virus emergence and spread. Of these mechanisms, the emergence of novel serotypes makes vaccination controversial despite its effectiveness, along with the vector’s ability to acquire the virus and successfully transmit it, spatio-temporal adaptation, and identification of new transmission routes. We also discussed the predisposition of the vertebrate and invertebrate hosts to contract the virus, as well as factors hijacking vector population activity and abundance. Indeed, the ecological dynamics of biting midges strongly influence the ability of the virus to be transmitted and to cause epizootic into parts of the world never before affected, helped by interactions between the pathogen and its host in addition to multiple routes deployed to escape defense barriers in the target infected cells. The points raised in this review can help inform aspects of interest for future studies that may elucidate factors to consider in monitoring and controlling the expansion of Culicoides-transmitted arboviruses.

The impact of Bluetongue virus on abortion in cattle, sheep and goats in Türkiye: 2012-2017

Bluetongue (BT) is a disease that affects domestic and wild ruminants, and it is caused by a virus called bluetongue virus (BTV) that is transmitted by Culicoides midges. Although clinical signs of BT are most apparent in sheep, BTV could induce abortion and birth defects in cattle, sheep and goats. BTV infection has been reported in Türkiye, but the role of BTV in cattle and small ruminant abortion cases in Türkiye remains uncertain. Therefore, this research aimed to fill this research gap by investigating the prevalence of BTV in cattle and small ruminant abortion cases. To investigate the frequency of BTV in ovine, caprine, and bovine foetuses, a total of 1718 foetuses were collected from different farms between 2012 and 2017. A one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect BTV RNA in aborted foetuses. BTV specific RNA was not detected in the analysed foetuses. To the best of my knowledge, this is the longest study that has investigated whether BTV infection has a role in cattle and small ruminant abortion cases in Türkiye. The findings of the study suggest that BTV infection has no impact on abortion in cattle, sheep and goats in the surveyed regions. The results of this study are limited only to the regions studied. Therefore, further epidemiological studies are needed to confirm the findings of this study.

Culicoides biting midges in urban areas of northern Spain.

Culicoides biting midges (Diptera: Ceratopogonidae) are known vectors of multiple arboviruses of veterinary and medical importance, as well as avian haemosporidian parasites. Despite their significant role as vectors, the distribution of Culicoides species in non-farmland habitats remains largely unknown. We conducted a study to assess the species community composition, abundance, and host feeding patterns of Culicoides biting midges in urban ecosystems in northern Spain. Light-baited suction traps were operated biweekly for 6 months across six urban landscapes (three cemeteries and three green areas). In total, 181 Culicoides specimens of 18 species were morphologically identified, with the Obsoletus complex being predominant (43.% of the total catches), followed by Culicoides kibunensis (21.5%). Culicoides midges peaked in early June in green areas and in May in cemeteries. Host feeding patterns revealed avian preferences (mainly Turdus spp.) in four Culicoides species. Despite the low overall number of specimens collected, a high diversity of biting midge species was recorded. These findings offer critical insights into Culicoides ecology and their interactions with hosts in urban settings, which are vital for disease surveillance and management strategies.

Multiple bloodmeals enhance dissemination of arboviruses in three medically relevant mosquito genera

BackgroundMosquitoes in nature may acquire multiple bloodmeals (BMs) over the course of their lifetime; however, incorporation of frequent feeding behavior in laboratory vector competence studies is rarely done. We have previously shown that acquisition of a second non-infectious BM can enhance early dissemination of Zika virus (ZIKV), dengue virus, and chikungunya virus in Aedes aegypti and ZIKV in Aedes albopictus mosquitoes, yet it is unknown if other taxonomically-diverse virus-vector pairings show a similar trend under a sequential feeding regimen.MethodsTo test this, we evaluated the impact of a second noninfectious BM on the vector competence of Aedes aegypti and Anopheles quadrimaculatus for Mayaro virus, Culex quinquefasciatus for West Nile virus, Aedes triseriatus for La Crosse virus, and Aedes aegypti for Oropouche virus (OROV). Female mosquitoes were fed BMs containing these viruses and half of them were given a second noninfectious BM at 3 or 4-days post infection. Mosquitoes were harvested at various time points and assayed for virus infection in bodies and disseminated infection in legs by performing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays.ResultsWe found that a second noninfectious BM had no impact on midgut infection rates but increased virus dissemination for all but one of the virus-vector pairings- Ae. aegypti and OROV. Unlike the other arboviruses under consideration, which are strictly mosquito-borne, biting midges (Culicoides spp.) serve as the main vector of OROV and this virus rarely disseminated to the mosquito leg tissue in our study.ConclusionsTaken together, our findings show that sequential blood feeding enhances virus dissemination across diverse arbovirus-vector pairings, representing three mosquito genera and virus families, but a second BM was insufficient to overcome a strong midgut virus escape barrier in a nonnatural virus–vector pairing.Graphical

Increased Virulence of Culicoides Midge Cell-Derived Bluetongue Virus in IFNAR Mice.

Bluetongue (BT) is a Culicoides midge-borne hemorrhagic disease affecting cervids and ruminant livestock species, resulting in significant economic losses from animal production and trade restrictions. Experimental animal infections using the α/β interferon receptor knockout IFNAR mouse model and susceptible target species are critical for understanding viral pathogenesis, virulence, and evaluating vaccines. However, conducting experimental vector-borne transmission studies with the vector itself are logistically difficult and experimentally problematic. Therefore, experimental infections are induced by hypodermic injection with virus typically derived from baby hamster kidney (BHK) cells. Unfortunately, for many U.S. BTV serotypes, it is difficult to replicate the severity of the disease seen in natural, midge-transmitted infections by injecting BHK-derived virus into target host animals. Using the IFNAR BTV murine model, we compared the virulence of traditional BHK cell-derived BTV-17 with C. sonorensis midge (W8) cell-derived BTV-17 to determine whether using cells of the transmission vector would provide an in vitro virulence aspect of vector-transmitted virus. At both low and high doses, mice inoculated with W8-BTV-17 had an earlier onset of viremia, earlier onset and peak of clinical signs, and significantly higher mortality compared to mice inoculated with BHK-BTV-17. Our results suggest using a Culicoides W8 cell-derived inoculum may provide an in vitro vector-enhanced infection to more closely represent disease levels seen in natural midge-transmitted infections while avoiding the logistical and experimental complexity of working with live midges.

Sampling of Culicoides with nontraditional methods provides unusual species composition and new records for southern Spain

BackgroundCulicoides midges have been well-studied in Spain, particularly over the last 20 years, mainly because of their role as vectors of arboviral diseases that affect livestock. Most studies on Culicoides are conducted using suction light traps in farmed environments, but studies employing alternative trapping techniques or focusing on natural habitats are scarce.MethodsIn the present study, we analyze Culicoides captured in 2023 at 476 sites in western Andalusia (southern Spain) using carbon dioxide-baited Biogents (BG)-sentinel traps across different ecosystems.ResultsWe collected 3,084 Culicoides midges (3060 females and 24 males) belonging to 23 species, including the new species Culicoides grandifovea sp. nov. and the first record of Culicoides pseudolangeroni for Europe. Both species were described with morphological and molecular methods and detailed data on spatial distribution was also recorded. The new species showed close phylogenetic relations with sequences from an unidentified Culicoides from Morocco (92.6% similarity) and with Culicoides kurensis. Culicoides imicola was the most abundant species (17.4%), followed by Culicoides grandifovea sp. nov. (14.6%) and Culicoides kurensis (11.9%). Interestingly, Culicoides montanus was the only species of the obsoletus and pulicaris species complexes captured, representing the first record of this species in southern Spain. A total of 53 valid Culicoides species have been reported in the area, with 48 already reported in literature records and 5 more added in the present study. Information on the flight period for the most common Culicoides species is also provided.ConclusionsTo the best of our knowledge, our study represents the most comprehensive effort ever done on nonfarmland habitats using carbon-dioxide baited suction traps for collecting Culicoides. Our data suggests that using carbon dioxide traps offers a completely different perspective on Culicoides communities compared with routinely used light traps, including the discovery of previously unrecorded species.Graphical

Survival and growth of M. perstans larvae in a human colon carcinoma cell line-based in vitro culture

Mansonella perstans infections are widespread in Sub-Saharan Africa and Central and South America and thus can be considered as the most prevalent parasite of man in tropical Africa. In contrast to the high prevalence, knowledge about the biology of this filarial nematode is restricted and no effective treatment regimens of this ivermectin-resistant parasite is lacking. An obstacle for the research is that M. perstans resides in body cavities and thus have been only rarely recovered during surgery or autopsy. Therefore, alternative methods like in vitro culture systems need to be implemented to decipher the nature of mansonellosis and effective drugs. Previously, we have established a monkey kidney epithelial cell-based in vitro culture for the maintenance of M. perstans infective larvae (L3) up to 77 days. However, no alternative for this culture system have been postulated to allow longer survival rates and development of adult worms in vitro. Thus, we aim to establish an alternative in vitro culture system for M. perstans L3. M. perstans L3 were isolated from engorged and laboratory reared Culicoides midges. The larvae were then cultured in Dulbecco's Modified Eagle Medium supplemented with either 10% foetal bovine serum (FBS), 10% newborn calf serum (NCS) or 1% bovine serum albumin (BSA) together with human colon carcinoma cells (HCT-8) as feeder cells. Survival and growth were recorded. We obtained that the 10% NCS culture condition was superior allowing long-term maintenance of M. perstans L3 for up to 100 days and boosted growth of the parasites for up to 5-folds compared to the initial size at culture inception. Although no moulting of the L3 into L4 or adult worms could be overserved, the human colon carcinoma cell-based in vitro culture provides an alternative platform to analyse M. perstans biology and screen for novel drugs against M. perstans.

Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

Bluetongue Risk Map for Vaccination and Surveillance Strategies in India.

Bluetongue virus (BTV, Sedoreoviridae: Orbivirus) causes an economically important disease, namely, bluetongue (BT), in domestic and wild ruminants worldwide. BTV is endemic to South India and has occurred with varying severity every year since the virus was first reported in 1963. BT can cause high morbidity and mortality to sheep flocks in this region, resulting in serious economic losses to subsistence farmers, with impacts on food security. The epidemiology of BTV in South India is complex, characterized by an unusually wide diversity of susceptible ruminant hosts, multiple vector species biting midges (Culicoides spp., Diptera: Ceratopogonidae), which have been implicated in the transmission of BTV and numerous co-circulating virus serotypes and strains. BT presence data (1997-2011) for South India were obtained from multiple sources to develop a presence/absence model for the disease. A non-linear discriminant analysis (NLDA) was carried out using temporal Fourier transformed variables that were remotely sensed as potential predictors of BT distribution. Predictive performance was then characterized using a range of different accuracy statistics (sensitivity, specificity, and Kappa). The top ten variables selected to explain BT distribution were primarily thermal metrics (land surface temperature, i.e., LST, and middle infrared, i.e., MIR) and a measure of plant photosynthetic activity (the Normalized Difference Vegetation Index, i.e., NDVI). A model that used pseudo-absence points, with three presence and absence clusters each, outperformed the model that used only the recorded absence points and showed high correspondence with past BTV outbreaks. The resulting risk maps may be suitable for informing disease managers concerned with vaccination, prevention, and control of BT in high-risk areas and for planning future state-wide vector and virus surveillance activities.

Experimental manipulation of nest temperature and relative humidity reduces ectoparasites and affects body condition of Blue Tits (Cyanistes caeruleus)

Many models predict changes in the distribution and incidence of diseases associated with climate change. However, studies exploring the effect of microclimatic changes on host–parasite relationships are scarce. In this study, we increased temperature or humidity in Eurasian Blue Tit Cyanistes caeruleus nestboxes during the breeding season, to analyse the effect of each variable on the abundance of ectoparasites and, also, on the body condition of the hosts. Temperature and humidity were experimentally increased on average by about 2°C and 15%, respectively. The abundance of blowfly Protocalliphora azurea pupae was significantly reduced in nests with increased temperature compared with control nests and those with increased humidity, and was also significantly reduced in nests with increased humidity compared with control nests. The abundance of mites Dermanyssus spp. was significantly reduced in nests with increased humidity. However, there was no significant effect of the experiment on the abundance of flea Ceratophyllus gallinae larvae, biting midges (Culicoides spp.) and blackflies (Simuliidae). The body condition of nestlings was lower in nests with increased humidity and slightly lower in heated nests. However, the body condition of adults was not significantly affected by the experiment. In addition, blowfly pupae and biting midge abundance were negatively related to the body condition of the nestlings, and biting midge abundance was also negatively related to the body condition of the adults. Overall, an increase in temperature and humidity reduces the abundance of ectoparasites, which suggests that these parasites are sensitive to sudden changes in the microclimate in which they develop. Moreover, these fluctuations negatively affect the body condition of Blue Tit nestlings despite the concurrent decrease of parasites in nests. In other words, the expected benefit to the nestlings from the reduction in parasites does not occur because the microclimate also adversely affects nestlings, probably affecting their heat exchange with the nest environment.

The Potential of Plant-Produced Virus-like Particle Vaccines for African Horse Sickness and Other Equine Orbiviruses.

African horse sickness is a devastating viral disease of equids. It is transmitted by biting midges of the genus Culicoides with mortalities reaching over 90% in naïve horses. It is endemic to sub-Saharan Africa and is seasonally endemic in many parts of southern Africa. However, outbreaks in Europe and Asia have occurred that caused significant economic issues. There are attenuated vaccines available for control of the virus but concerns regarding the safety and efficacy means that alternatives are sought. One promising alternative is the use of virus-like particles in vaccine preparations, which have the potential to be safer and more efficacious as vaccines against African horse sickness. These particles are best made in a complex, eukaryotic system, but due to technical challenges, this may cause significant economic strain on the developing countries most affected by the disease. Therefore, this review also summarises the success so far, and potential, of recombinant protein expression in plants to reduce the economic strain of production.

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.

Seasonal change and influence of environmental variables on host-seeking activity of the biting midge Culicoides sonorensis at a southern California dairy, USA

BackgroundAs a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk.MethodsThe diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fisher’s exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity.ResultsHost-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking.ConclusionsThis study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.Graphical

Variable gut pH as a potential mechanism of tolerance to Bacillus thuringiensis subsp. israelensis toxins in the biting midge Culicoides sonorensis.

Toxins of Bacillus thuringiensis subsp. israelensis (Bti) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi-aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti-based commercial insecticide and its active ingredient in larval Culicoides sonorensis. A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti. The lethal concentration to kill 90% (LC90) of larvae ofthe commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.

Comparative genomics of a novel Erwinia species associated with the Highland midge (Culicoides impunctatus).

Erwinia (Enterobacterales: Erwiniaceae) are a group of cosmopolitan bacteria best known as the causative agents of various plant diseases. However, other species in this genus have been found to play important roles as insect endosymbionts supplementing the diet of their hosts. Here, I describe Candidatus Erwinia impunctatus (Erwimp) associated with the Highland midge Culicoides impunctatus (Diptera: Ceratopogonidae), an abundant biting pest in the Scottish Highlands. The genome of this new Erwinia species was assembled using hybrid long and short read techniques, and a comparative analysis was undertaken with other members of the genus to understand its potential ecological niche and impact. Genome composition analysis revealed that Erwimp is similar to other endophytic and ectophytic species in the genus and is unlikely to be restricted to its insect host. Evidence for an additional plant host includes the presence of a carotenoid synthesis operon implicated as a virulence factor in plant-associated members in the sister genus Pantoea. Unique features of Erwimp include several copies of intimin-like proteins which, along with signs of genome pseudogenization and a loss of certain metabolic pathways, suggests an element of host restriction seen elsewhere in the genus. Furthermore, a screening of individuals over two field seasons revealed the absence of the bacteria in Culicoides impunctatus during the second year indicating this microbe-insect interaction is likely to be transient. These data suggest that Culicoides impunctatus may have an important role to play beyond a biting nuisance, as an insect vector transmitting Erwimp alongside any conferred impacts to surrounding biota.

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

Population genetics of the biting midge Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) from Thailand and its genetic relationships with global populations

Population genetics of the biting midge Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) from Thailand and its genetic relationships with global populations

A filarial parasite potentially associated with the health burden on domestic chickens in Japan

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.

Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

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.