Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep.

Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of endocrine glands and the pathogenesis it induces. The aim of the study was to assess the BTV load, antigen distribution in the tissue of the pituitary, thyroid as well as adrenal glands and associated histopathological consequences. BTV antigens were localized using immunohistochemistry in the thyroid's epithelial cells, zona fasciculata and zona reticularis cells and the anterior pituitary epithelial cells. The real-time PCR portrayed the high viral load in adrenals at 7th days postinoculation (DPI) and in thyroid and pituitary glands at 15th DPI. Serum examination revealed variation in the T-3 and T-4 of infected animals in comparison to the control group. Caspase-3 immunolocalization revealed BTV-1 induces apoptosis in the affected cells of endocrine gland of infected animals. Further, this study signifies the tropism of BTV in the novel sites (endocrine glands) of the host that might be one of the reasons for the poor performance of infected animals.

Sero-epidemiology of bluetongue in ruminants raised on private holdings in North-Western Pakistan.

This study investigated the sero-epidemiology of bluetongue in ruminants in North-Western Pakistan. A total of 3,173 serum samples were collected from small (n = 1,651) and large (n = 1,522) ruminants being reared by farmers in 14 districts. Antibodies to bluetongue virus (BTV) were detected using competitive ELISA. The overall prevalence of BTV antibodies was 65%. A significant association (P < 0.05) between the prevalence of BTV antibodies and the risk factors including sex, species, age, area, husbandry practices and breed was shown by univariate analysis. In multivariate analysis, the seroprevalence was 6.5 (95% CL = 3.7-11.4), 5.9 (95% CL = 3.8-9.4) and 2.4 (95% CL = 1.5-3.7) times higher in buffaloes, cattle and goats than sheep, respectively. The seroprevalence was 1.4 (95% CL = 1.1-1.7) times higher in local breeds than in cross/exotic breeds. The seroprevalence was 1.6 (95% CL = 1.1 to 2.3) times higher in sedentary animals than in nomadic animals. The seroprevalence was significantly associated with age. Further work is required to determine the BTV serotypes prevalent in the study area for effective control of the disease.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo.

Segmented RNA viruses are a taxonomically diverse group that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during coinfection of a host by a process termed "reassortment." Reassortment enables rapid evolutionary change, but where transmission involves a biological arthropod vector, this change is constrained by the selection pressures imposed by the requirement for replication in two evolutionarily distant hosts. In this study, we use an in vivo, host-arbovirus-vector model to investigate the impact of reassortment on two phenotypic traits, virus infection rate in the vector and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV comprises 10 linear segments of dsRNA, and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4, and BTV-8) were isolated from naturally infected ruminants in Europe and ancestral/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to replicate in vector Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the ancestral virus strains, which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCE Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur. Utilizing a representative mammalian host-insect vector infection and transmission model, we provide direct evidence of this phenomenon in closely related ancestral and reassortant strains of BTV. Our results demonstrate that efficient infection of Culicoides observed for one of three ancestral BTV strains was also evident in two reassortant strains that had subsequently emerged in the same ecosystem.

Sialic Acid Binding Sites in VP2 of Bluetongue Virus and Their Use during Virus Entry.

Bluetongue virus (BTV), a member of the Orbivirus genus, is transmitted by biting midges (gnats, Culicoides sp.) and is one of the most widespread animal pathogens, causing serious outbreaks in domestic animals, particularly in sheep, with high economic impact. The non-enveloped BTV particle is a double-capsid structure of seven proteins and a genome of 10 double-stranded RNA segments. Although the outermost spike-like VP2 acts as the attachment protein during BTV entry, no specific host receptor has been identified for BTV. Recent high-resolution cryo-electron (cryoEM) structures and biological data have suggested that VP2 may interact with sialic acids (SAs). To confirm this, we have generated protein-based nanoparticles displaying multivalent VP2 and used them to probe glycan arrays. The data show that VP2 binds α2,3-linked SA with high affinity but also binds α2,6-linked SA. Further, Maackia amurensis lectin II (MAL II) and Sambucus nigra lectin (SNA), which specifically bind α2,3-linked and α2,6-linked SAs, respectively, inhibited BTV infection and virus growth in susceptible sheep cells while SNA alone inhibited virus growth in Culicoides-derived cells. A combination of hydrogen deuterium exchange mass spectrometry and site-directed mutagenesis allowed the identification of the specific SA binding residues of VP2. This study provides direct evidence that sialic acids act as key receptor for BTV and that the outer capsid protein VP2 specifically binds SA during BTV entry in both mammalian and insect cells. IMPORTANCE To date no receptor has been assigned for non-enveloped bluetongue virus. To determine if the outermost spike-like VP2 protein is responsible for host cell attachment via interaction with sialic acids, we first generated a protein-based VP2-nanoparticle, for the multivalent presentation of recombinant VP2 protein. Using nanoparticles displaying VP2 to probe a glycan array, we identified that VP2 binds both α2,3-linked and α2,6-linked sialic acids. Lectin inhibitors targeting both linkages of sialic acids showed strong inhibition to BTV infection and progeny virus production in mammalian cells; however the inhibition was only seen with the lectin targeting α2,6-linked sialic acid in insect vector cells. In addition, we identified the VP2 sialic acid binding sites in the exposed tip domain. Our data provides direct evidence that sialic acids act as key receptors for BTV attachment and entry in to both mammalian and insect cells.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Chimaeric plant-produced bluetongue virus particles as potential vaccine candidates.

Bluetongue virus (BTV) causes bluetongue disease in ruminants and sheep. The current live attenuated and inactivated vaccines available for prevention pose several risks, and there is thus a need for vaccines that are safer, economically viable, and effective against multiple circulating serotypes. This work describes the development of recombinant virus-like particle (VLP) vaccine candidates in plants, which are assembled by co-expression of the four BTV serotype 8 major structural proteins. We show that substitution of a neutralising tip domain of BTV8 VP2 with that of BTV1 VP2 resulted in the assembly of VLPs that stimulated serotype-specific antibodies as well as virus-specific neutralising antibodies.

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.

An Agent-Based Model of Biting Midge Dynamics to Understand Bluetongue Outbreaks.

Bluetongue (BT) is a well-known vector-borne disease that infects ruminants such as sheep, cattle, and deer with high mortality rates. Recent outbreaks in Europe highlight the importance of understanding vector-host dynamics and potential courses of action to mitigate the damage that can be done by BT. We present an agent-based model, entitled 'MidgePy', that focuses on the movement of individual Culicoides spp. biting midges and their interactions with ruminants to understand their role as vectors in BT outbreaks, especially in regions that do not regularly experience outbreaks. The results of our sensitivity analysis suggest that midge survival rate has a significant impact on the probability of a BTV outbreak as well as its severity. Using midge flight activity as a proxy for temperature, we found that an increase in environmental temperature corresponded with an increased probability of outbreak after identifying parameter regions where outbreaks are more likely to occur. This suggests that future methods to control BT spread could combine large-scale vaccination programs with biting midge population control measures such as the use of pesticides. Spatial heterogeneity in the environment is also explored to give insight on optimal farm layouts to reduce the potential for BT outbreaks.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Molecular identification of Culicoides oxystoma and Culicoides actoni vectors of bluetongue virus.

Bluetongue is a non-contagious viral disease causing significant economic losses throughout the world. The bluetongue vectors Culicoides oxystoma and Culicoides actoni, which play a significant role in the transmission of various pathogens, are distributed across different geographical realms. Adults are minute in size with wide phenotypic variation, so morphology-based species identification is severely constrained by preparatory time and shortage of taxonomic expertise. To make the identification process rapid and effective, a specific primer was designed for the identification of C. actoni based on the multiple sequence alignment of ITS1 sequences of 11 Culicoides species. Along with this, a refined version of existing C. oxystoma specific primer was proposed. The primer sets distinguished C. oxystoma and C. actoni from a pooled sample consisting of other Culicoides species as well as closely related genera such as Forcipomyia and Alluaudomyia. Our findings suggest that the primers were species specific, sensitive and have potential to discriminate vector species C. oxystoma and C. actoni from pooled samples. To the best of our knowledge, these are the first ITS1 sequences generated and submitted in GenBank for Culicoides innoxius, Culicoides shortti, Culicoides palpifer and Culicoides anophelis and the first for Culicoides peregrinus, Culicoides fulvus and C. actoni from India.

The use of path analysis to determine effects of environmental factors on the adult seasonality of Culicoides (Diptera: Ceratopogonidae) vector species in Spain.

Culicoides biting midges (Diptera: Ceratopogonidae) are the main vectors of livestock diseases such as bluetongue (BT) which mainly affect sheep and cattle. In Spain, bluetongue virus (BTV) is transmitted by several Culicoides taxa, including Culicoides imicola, Obsoletus complex, Culicoides newsteadi and Culicoides pulicaris that vary in seasonality and distribution, affecting the distribution and dynamics of BT outbreaks. Path analysis is useful for separating direct and indirect, biotic and abiotic determinants of species' population performance and is ideal for understanding the sensitivity of adult Culicoides dynamics to multiple environmental drivers. Start, end of season and length of overwintering of adult Culicoides were analysed across 329 sites in Spain sampled from 2005 to 2010 during the National Entomosurveillance Program for BTV with path analysis, to determine the direct and indirect effects of land use, climate and host factor variables. Culicoides taxa had species-specific responses to environmental variables. While the seasonality of adult C. imicola was strongly affected by topography, temperature, cover of agro-forestry and sclerophyllous vegetation, rainfall, livestock density, photoperiod in autumn and the abundance of Culicoides females, Obsoletus complex species seasonality was affected by land-use variables such as cover of natural grassland and broad-leaved forest. Culicoides female abundance was the most explanatory variable for the seasonality of C. newsteadi, while C. pulicaris showed that temperature during winter and the photoperiod in November had a strong effect on the start of the season and the length of overwinter period of this species. These results indicate that the seasonal vector-free period (SVFP) in Spain will vary between competent vector taxa and geographic locations, dependent on the different responses of each taxa to environmental conditions.

Development and evaluation of gold nanoprobe based lateral flow device for rapid and sensitive serodetection of Bluetongue in sheep.

Bluetongue (BT) disease is a viral, insect borne, noncontagious illness of small ruminants caused by Orbivirus, impacting huge economic loss worldwide. The existing BT diagnostic techniques are costly, time-consuming and require both specialized equipment and also skilled personnel. So there is need to develop a rapid, sensitive, on site detection assay for diagnosis of BT. This study utilized secondary antibody derivatized Gold nanoprobes for rapid and sensitive detection of BT over lateral flow device (LFD). The detection limit for this assay was found 1.875 µg of BT IgG/ml and a comparison between LFD and indirect ELISA was performed and the sensitivity and specificity was found at 96% and 99.23%, respectively, with observed kappa value of 0.952. This developed LFD may therefore offer a quick, affordable and accurate diagnosis of BT disease at the field level.

Assessing the export trade risk of bluetongue virus serotypes 4 and 8 in France.

Bluetongue (BT) causes an economic loss of $3 billion every year in the world. After two serious occurrences of BT (bluetongue virus [BTV] occurrence in 2006 and 2015), France has been controlling for decades, but it has not been eradicated. As the largest live cattle export market in the world, France is also one of the major exporters of breeding animals and genetic materials in the world. The biosafety of its exported cattle and products has always been a concern. The scenario tree quantitative model was used to analyze the risk of BTV release from French exported live cattle and bovine semen. The results showed that with the increase in vaccination coverage rates, the risk decreased. If the vaccine coverage is 0%, the areas with the highest average risk probability of BTV-4 and BTV-8 release from exported live cattle were Haute-Savoie and Puy-de-Dôme, and the risk was 2.96 × 10-4 and 4.25 × 10-4 , respectively. When the vaccine coverage was 90%, the risk probability of BTV-4 and BTV-8 release from exported live cattle was 2.96 × 10-5 and 4.24 × 10-5 , respectively. The average probability of BTV-8 release from bovine semen was 1.09 × 10-10 . Sensitivity analysis showed that the probability of false negative polymerase chain reaction (PCR) test and the probability of BT infection in the bull breeding station had an impact on the model. The identification of high-risk areas and the discovery of key control measures provide a reference for decision makers to assess the risk of French exports of live cattle and bovine semen.

The investigation of Culicoides (Diptera: Ceratopogonidae) species and Bluetongue virus and Schmallenberg virus in Northwest Türkiye.

Culicoides biting midges (Diptera: Ceratopogonidae) act as mechanical and biological vectors of arboviruses and are crucial in the global spread of these viruses. This study investigated the diversity of distribution of Culicoides species and the presence of Bluetongue virus (BTV) and Schmallenberg virus (SBV) in Tekirdag province in Northwest Türkiye. The fourteen Culicoides species, such as Culicoides newsteadi, Culicoides schultzei, Culicoides nubeculosus comp., Culicoides punctatus, Culicoides circumscriptus, Culicoides obsoletus comp., Culicoides gejgelensis, Culicoides festivipennis, Culicoides longipennis, Culicoides spp., Culicoides pulicaris, Culicoides picturatus, Culicoides odiatus, Culicoides kurensis, and Culicoides flavipulicaris, were detected. Culicoides newsteadi, C. odiatus, and C. pulicaris were the most abundant species. Phylogenetic analyses of Culicoides species' ITS-1 gene region were performed. A pool of C. festivipennis was positive for SBV RNA, while the BTV genomic materials was not found in the qPCR analysis. This is the first report of the presence/detection of SBV in Culicoides species in Türkiye. The survey of bioecological and epizootiological aspects of vector species is essential in implementing effective control measures for arboviral infections.

Bluetongue virus infection in cattle: serosurvey and its associated risk factors.

Bluetongue virus (BTV) is a vector-borne virus that primarily affects sheep. However, the disease is usually asymptomatic in cattle without obvious clinical signs related to BTV infection. Although there is evidence of BTV antibodies through serology in Egypt, it is still unknown whether Egyptian cattle have ever been exposed to the virus in the north or south of the country. The study's aims were to determine the seroprevalence of BTV and evaluate the potential risk factors for BTV infection in cattle in Egypt. We used a competitive ELISA to screen 690 healthy cattle for BTV-specific immunoglobulin G (IgG) antibodies in four governorates in Egypt. A total seroprevalence of BTV antibodies in examined cattle was 51.47%, 95%CI: 48.01-55.45. The odds of BTV seropositivity were higher in Aswan (OR=1.30, 95%CI: 0.71-2.36), females (OR=3.29, 95%CI: 1.87-5.79), and elder cattle >8 years (OR=12.91, 95%CI: 6.63-25.13). Moreover, cattle contacted with other animals (OR=1.40, 95%CI: 0.94-2.10), with history of abortion (OR=4.88, 95%CI: 3.14-7.59), and those living with presence of insects (OR=12.34, 95%CI: 8-19.30) were more likely to be infected with bluetongue (BT). To effectively predict and respond to a potential BTV outbreak in Egypt, surveillance for BTV infection should be expanded to cover other susceptible ruminants and the range of the insect vectors.

Comparison of machine learning models for bluetongue risk prediction: a seroprevalence study on small ruminants.

BACKGROUND: Bluetongue (BT) is a disease of concern to animal breeders, so the question on their minds is whether they can predict the risk of the disease before it occurs. The main objective of this study is to enhance the accuracy of BT risk prediction by relying on machine learning (ML) approaches to help in fulfilling this inquiry. Several risk factors of BT that affect the occurrence and magnitude of animal infection with the virus have been reported globally. Additionally, risk factors, such as sex, age, species, and season, unevenly affect animal health and welfare. Therefore, the seroprevalence study data of 233 apparently healthy animals (125 sheep and 108 goats) from five different provinces in Egypt were used to analyze and compare the performance of the algorithms in predicting BT risk. RESULTS: Logistic regression (LR), decision tree (DT), random forest (RF), and a feedforward artificial neural network (ANN) were used to develop predictive BT risk models and compare their performance to the base model (LR). Model performance was assessed by the area under the receiver operating characteristics curve (AUC), accuracy, true positive rate (TPR), false positive rate (FPR), false negative rate (FNR), precision, and F1 score. The results indicated that RF performed better than other models, with an AUC score of 81%, ANN of 79.6%, and DT of 72.85%. In terms of performance and prediction, LR showed a much lower value (AUC = 69%). Upon further observation of the results, it was discovered that age and season were the most important predictor variables reported in classification and prediction. CONCLUSION: The findings of this study can be utilized to predict and control BT risk factors in sheep and goats, with better diagnostic discrimination in terms of accuracy, TPR, FNR, FPR, and precision of ML models over traditional and commonly used LR models. Our findings advocate that the implementation of ML algorithms, mainly RF, in farm decision making and prediction is a promising technique for analyzing cross-section studies, providing adequate predictive power and significant competence in identifying and ranking predictors representing potential risk factors for BT.

A survey of bluetongue infection in one-humped camels (Camelus Dromedarius); seroprevalence and risk factors analysis.

Bluetongue (BT) is an insect-borne, non-contagious viral disease which affects domestic ruminants including camels and is transmitted by Culicoides spp. Clinical symptoms of BT are typically seen in sheep, although subclinical BT infections are mostly seen in cattle, goats, and camelids. The goal of the present study was to evaluate the sero-prevalence of Bluetongue virus (BTV) in camels from some governorates in Egypt's southern and northern regions, as well as the infection's potential risk factors. During 2020-2021, a cross sectional study was conducted to screen presence of anti-BTV antibodies in 400 serum samples, which were collected randomly from camels, examined using competitive enzyme-linked immunosorbent assay (cELISA). The sera of 102 out of 400 camels tested positive for BTV, representing a frequency of 25.5%. Moreover, the odds of sero-positivity were higher among camels living in Aswan (OR = 5.33, 95%CI: 2.35-12.11), especially in females (OR = 2.63, 95%CI = 1.44-4.09) during summer season (OR = 2.40, 95%CI = 1.20-4.81). Furthermore, the probability of getting BTV infection increased when camels were exposed to the insect vectors (OR = 1.63, 95%CI = 0.87-3.09). The high prevalence of BTV in camels in several Egyptian regions highlights the need for more epidemiological investigations of BTV infection in other ruminant species in order to better control BT disease in these regions.

Blood meal analysis of Culicoides species associated with livestock in West Bengal, India.

Knowledge gaps exist on the feeding pattern and host range of bluetongue virus vectors, Culicoides species, associated with livestock in India. Adult midges were trapped with ultraviolet light traps at 13 household farms adjacent to human biotope. Host DNA was isolated from individual females (n = 101; blood engorged-82, gravid-4 and parous-15) and subjected to PCR amplification targeting CytB and 16S rRNA gene fragments followed by sequencing of amplified DNA samples. However, DNA sequences from only 71 individuals (70.3%) comprising of 10 Culicoides species were obtained. Blood meal analysis revealed at least 10 species that fed on five mammalian hosts including humans, but surprisingly none tested positive for birds. Results revealed that Culicoides innoxius tested positive for four not previously recognized species indicating a potential role as a vector species. Likewise, Culicoides shortti and Culicoides hegneri preferred goat and cattle respectively as hosts, whereas Culicoides palpifer preferred cattle along with buffalo as hosts, which is being reported for the first time. This is the first document on DNA-based blood meal identification and feeding preference of Culicoides midges associated with livestock in India.