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Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface., (© 2020 Blackwell Verlag GmbH.)

Bluetongue virus serotype 8 (BTV-8) caused an epizootic in Europe in 2006/09. Transplacental transmission of BTV-8 was demonstrated leading to abortions, congenital malformations or nervous clinical signs in newborn calves. BTV-8 re-emerged in France in 2015. Although the re-emergent strain is nearly genetically identical to the one that had circulated in 2006/2009, it has caused very few clinical cases. However, from mid-December 2018 to April 2019, cases of calves with congenital malformations or displaying nervous clinical signs occurred in some departments (French administrative unit) in mainland France. Blood samples from these animals were sent to local laboratories, and the positive ones were confirmed at the French Bluetongue reference laboratory (BT-NRL). Out of 580 samples found positive at the local laboratories, 544 were confirmed as RT-PCR BTV-8 positive. The 36 samples found positive in the local laboratories and negative in the BT-NRL were all at the limit of RT-PCR detection. Hundred eighty-eight of the confirmed samples were also tested for the presence of Schmallenberg virus (SBV) and bovine virus diarrhoea virus (BVDV) infection: 4 were found positive for BVDV and none for SBV. The main clinical signs recorded for 244 calves, for which a reporting form was completed by veterinarians, included nervous clinical signs (81%), amaurosis (72%) and decrease/ no suckling reflex (40%). Hydranencephaly and microphthalmia were reported in 19 calves out of 27 in which a necropsy was practiced after death or euthanasia. These results indicate that the re-emergent strain of BTV-8 can cross the transplacental barrier and cause congenital malformations or nervous clinical signs in calves., (© 2019 Blackwell Verlag GmbH.)

Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV-28. Phylogenetic analysis of BTV-28 segment 2 (Seg-2) showed that it is related to Seg-2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%-66% identity in nucleotide sequences (nt) and 59%-62% in amino acid (aa) sequences of BTV VP2. BTV-28 Seg-6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg-5 was most closely related to a South African BTV-4 strain, and all other segments showed close similarity to BTV-26. Experimental infection by injection of 6-month-old ewes caused clinical signs in all injected animals, lasting from 2 to 3 days to several weeks post-infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV-injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact-infected animals. These findings demonstrate that BTV-28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts., (© 2019 Blackwell Verlag GmbH.)

The outbreak of bluetongue virus (BTV) serotype 8 (BTV-8) during 2006-2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV-8 strain re-emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV-8 strain alongside the re-emerging BTV-8 strain isolated from France in 2017. Two groups of eight BTV-naïve British mule sheep were inoculated with 5.75 log 10 TCID 50 /ml of either BTV-8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5-9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0-8.8 log 10 genome copies/ml) than the re-emerging BTV-8 strain (2.9-7.9 log 10 genome copies/ml). All infected sheep developed BTV-specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV-8-inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV-8. In Culicoides sonorensis feeding on the sheep over the period 7-12 dpi, vector competence was significantly higher for the 2007 strain than the re-emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re-emerging strain. However, one sheep infected with the re-emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re-emerging BTV-8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry., (© 2019 The Pirbright Institute. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides-borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind-blown dissemination of infected midges. Here, we report the first identification of BTV-3 in Sardinia, Italy. BTV-3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV-3 SAR2018 was isolated on cell culture. BTV-3 SAR2018 sequence and partial sequences obtained by next-generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99-100% of nucleotide identity) to BTV-3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

In November 2017, a 15-day-old calf located in France (Haute-Savoie department) was found positive for bluetongue virus (BTV) RNA by RT-PCR. Laboratory investigations allowed the isolation and identification of the serotype: BTV-4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV-4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV-4 has been introduced to mainland France from Corsica or Italy where BTV-4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV-4 in mainland France., (© 2018 Blackwell Verlag GmbH.)

Bluetongue (BT), caused by bluetongue virus (BTV), is a vector-borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross-sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme-linked immunosorbent assay (cELISA) and real-time polymerase chain reaction (RT-PCR). Sera (n = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n = 300), Mastung (n = 201), Killa Saifullah (n = 75) and Kech (n = 300). None of the study herds (n = 97) were seronegative for BTV, and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n = 414/876, 95% CI = 43.92%-50.63%). A higher percentage of goats (50.87%, 95% CI = 45.99%-55.73%) were seropositive for anti-VP7 immunoglobulins (IgG) than sheep (44.21%, 95% CI = 39.81%-48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ 2  = 16.84, p = .01), parity (χ 2  = 23.66, p = .00) and presence of vector (χ 2  = 2.63, p = .10), whereas for sheep, it was associated with breed type (χ 2  = 13.80, p = .01) and parity (χ 2  = 53.40, p = .00). Serotype 8 was the most prevalent (26.82%, 95% CI = 14.75%-43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI = 1.91%-21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan., (© 2018 Blackwell Verlag GmbH.)

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population., (© 2017 Blackwell Verlag GmbH.)

In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV-25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with bluetongue infection, a scenario described in animals infected with atypical BTV strains., (© 2018 Blackwell Verlag GmbH.)

In 2014, a new bluetongue virus serotype 4 (BTV-4) strain was detected in southern Greece and spread rapidly throughout the Balkan Peninsula and adjacent countries. Within half a year, more than 7,068 outbreaks were reported in ruminants, particularly in sheep. However, the reported morbidity and case fatality rates in ruminants varied. The pathogenesis of a Bulgarian BTV-4 strain isolated from sheep during the BTV-4 epizootic was studied in different species. Therefore, four sheep, three goats and three cattle were experimentally infected with the isolate BTV-4/BUL2014/15 and monitored for clinical signs up to several weeks. Serum and whole-blood samples were collected at regular intervals and subjected to serological and virological analyses. In this context, BTV-4-specific real-time RT-PCR assays were developed. The infection kinetics were similar to those known for other traditional BTV serotypes, and only mild BT-like clinical signs were observed in goats and sheep. In cattle, no obvious clinical signs were observed, except a transient increase in body temperature. The study results contrast with the severe clinical signs reported in sheep experimentally infected with an African BTV-4 strain and with the reports of BT-like clinical signs in a considerable proportion of different ruminant species infected with BTV-4 in the Balkan region and Italy. The discrepancies between the results of these animal trials and observations of BTV-4 infection in the field may be explained by the influence of various factors on the manifestation of BT disease, such as animal breed, fitness and virus strain, as described previously., (© 2017 Blackwell Verlag GmbH.)

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV-26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV-27v01-v03) were recently detected in asymptomatic goats in Corsica, France, 2014-2015. Molecular characterization revealed genetic differences between the three variants. Therefore, in vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV-naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV-RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV-Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole-blood was possible at the peak of viremia until 49 dpi. Moreover, detection of BTV-27v02-RNA and Ab in one contact goat indicated that-similar to BTV-26-at least one of three BTV-27 variants may be transmitted by contact between goats. In the field, BTV-27 RNA can be detected up to 6 months in the whole-blood of BTV-27-infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV-27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT-like clinical signs. In summary, the phenotypes observed for BTV-27v01-v03 phenotypes correspond to a mixture of characteristics known for BTV-25 and 26., (© 2017 Blackwell Verlag GmbH.)

Bluetongue virus serotype 8 (BTV-8) re-emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV-8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT-PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re-emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDVet) and, when enough serum was available, ELISA-positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re-emergence (N = 14 of 511), significantly more on the following year (N = 17 of 257), and eight animals (N = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N = 12 of 150), and in winter 2015/2016 in three of them (N = 21 of 555), where seven animals (N = 154) seroconverted over 2015. These results suggest that BTV-8 may have spread at low levels before the re-emergence, even in areas considered virus-free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT-PCR., (© 2017 Blackwell Verlag GmbH.)

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador., (© 2017 Blackwell Verlag GmbH.)

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe., (© 2017 Blackwell Verlag GmbH.)

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state., (© 2017 Blackwell Verlag GmbH.)

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks., (© 2016 Blackwell Verlag GmbH.)

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously., (© 2016 Blackwell Verlag GmbH.)

Bluetongue is endemic in China, and Bluetongue virus (BTV) strains belonging to eight different serotypes (BTV-1, BTV-2, BTV-3, BTV-4, BTV-9, BTV-12, BTV-15 and BTV-16) had been isolated between 1996 and 1997. However, there has been a long pause in investigating the epidemiology of BTV infection since then. During 2012-2014, eight BTV strains belonging to serotypes 5, 7 and 24 were isolated for the first time in Yunnan and Guangdong provinces from the blood of sentinel animals. Phylogenetic analyses of genome segment 2 of these Chinese BTV strains grouped them into nucleotypes E, F and A, respectively, along with the reference strains of the same serotype. For each serotype, Chinese strains cluster together closely to form a China's sublineage. In addition, these strains were most closely related to strains from Africa, indicating that they may share a recent common ancestry with African strains. To our knowledge, this is the first time that BTV-5, BTV-7 and BTV-24 strains have been isolated in South-East Asia. These data will be beneficial for understanding the BTV epidemiology and improving diagnostic assays and control measures against bluetongue in China and its neighbouring countries in the Asia-Pacific region., (© 2016 Blackwell Verlag GmbH.)

Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory., (© Her Majesty the Queen in Right of Canada (2015). Reproduced with the permission of the Minister of Health of Canadian Food Inspection Agency.)

One hundred and twenty-six blood samples were collected from healthy sheep and goats in Xinjiang, China, during July 2014. Seventy-three samples (57.93%) were bluetongue virus (BTV) serology-positive, and 39 samples (30.95%) were BTV NS1 gene-positive. BTV strain XJ1407 was isolated from the blood of BTV NS1 gene-positive animals and sequenced. Analysis of its genome sequence suggests that XJ1407 is a novel BTV serotype., (© 2016 Blackwell Verlag GmbH.)

An epidemiological study was carried out to determine the spatial-temporal trends and risk factors potentially involved in the seropositivity to bluetongue virus (BTV) in hunting areas with presence of red deer (Cervus elaphus). A total of 60 of 98 (61.2%; CI95% : 51.6-70.9) hunting areas sampled presented at least one seropositive red deer. Antibodies against BTV were detected in juvenile animals during the hunting seasons 2007/2008 to 2013/2014 in 15 of 98 (15.3%) hunting areas, which indicates an uninterrupted circulation of BTV in this period. A multivariate logistic regression model showed that the red deer density at hunting area level (>22 individuals/km(2) ), the annual abundance of Culicoides imicola (>1.4 mosquitoes/sampling) and the goat density at municipality level (>24.1 individuals/km(2) ) were factors significantly associated with BTV seropositivity in hunting areas. Control measures against BTV in the studied area include vaccination programmes in wild and domestic ruminants, movement control in areas with high densities and abundance of red deer and C. imicola, respectively. Considering the potential risk of BTV re-emergence, red deer should be included in the BT surveillance programmes in regions where these species share habitats with livestock., (© 2014 Blackwell Verlag GmbH.)

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern., (© 2016 Blackwell Verlag GmbH.)

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations., (© 2014 Blackwell Verlag GmbH.)

In 2007, bluetongue virus (BTV) was introduced to both Denmark (DK) and the United Kingdom (UK). For this reason, simulation models were built to predict scenarios for future incursions. The DK and UK models have a common description of within-herd dynamics, but differ greatly in their descriptions of between-herd spread, one using an explicit representation of vector dispersal, the other a transmission kernel. Here, we compare model predictions for the dynamics of bluetongue in the UK, based on the 2007 incursion and vaccination rollout in 2008. We demonstrate how an agent-based model shows greater sensitivity to the level of vaccine uptake and has lower variability compared with a kernel-based model. However, a model using a transmission kernel requires less detailed data and is often faster., (© 2014 Blackwell Verlag GmbH.)

The immunogenicity of two inactivated bluetongue virus serotype 8 (BTV-8) vaccines was evaluated in 880 cattle under field conditions. The effect of selected factors on vaccine performance was also analysed at the herd and animal levels (vaccine, herd size and production, age, sex, time interval between vaccination and blood sampling and veterinary training). The immunogenicity elicited by vaccination with the two vaccines was monitored with the aid of a competitive enzyme-linked immunosorbent assay (c-ELISA) and serum neutralization test (SNT). To investigate whether the selected factors influenced seroconversion at the herd and animal levels, a multilevel logistic regression model developed in a mixed model was applied. Of the 880 cattle vaccinated, 76.0% yielded BTV c-ELISA antibodies, whereas only 25.0% seroconverted based on SNT. Type of vaccine (odds ratio [OR] 4.5; 95% confidence interval [CI], 2.2-9.0 for SNT and OR 3.5; 95% CI, 2.1-5.9 for c-ELISA), veterinary training in vaccine administration (OR 8.1; 95% CI, 4.7-14.1 for SNT and OR 2.4; 95% CI, 1.3-4.2 for c-ELISA), animal age (OR 1.4; 95% CI, 1.1-1.8 for SNT and OR 1.7; 95% CI, 1.4-2.1 for c-ELISA) and days between first vaccine administration and blood collection (OR 1.9; 95% CI, 1.1-3.1 for SNT and OR 2.6; 95% CI, 1.7-3.8 for c-ELISA) were the major factors affecting vaccine performance under field conditions. This is the first study to use multilevel logistic regression in the evaluation of selected risk factors affecting BTV-8 vaccine performance in cattle., (© 2014 Blackwell Verlag GmbH.)

Since 2000, French Corsica Island has been exposed to the emergence of three different BT virus (BTV) serotypes: serotype 2 in 2000 and 2001, serotype 4 in 2003 and serotype 16 in 2004. Between 2005 and August 2013, no outbreaks have been reported in the French Island. At the beginning of September 2013, sheep located in the south of the island showed clinical signs suggestive of BTV infection. Laboratory analyses identified the virus as BTV serotype 1. Phylogenetic studies showed that the sequences of this strain are closely related to the BTV-1 strain that was circulating in the Mediterranean basin and in Sardinia in 2012., (© 2014 Blackwell Verlag GmbH.)

Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype., (© 2013 Blackwell Verlag GmbH.)

Bluetongue virus (BTV) causes disease mainly in sheep, but can be transmitted via other domestic and wild ruminants, resulting in pecuniary burden and trade restrictions. Segmented genome with the possibility of reassortment, existence of 26 serotypes, geographical restriction in the distribution of many of the serotypes, use of live attenuated vaccines and the lack of complete sequences of viruses isolated from several parts of the globe have complicated our understanding of the origin, movement and distribution of BTV. Recent efforts in genome sequencing of several strains have helped in better comprehending BTV epidemiology. In an effort to contribute to the genetic epidemiology of BTV in India, we report the isolation and complete genome sequencing of a BTV serotype 12 virus (designated NMO1). This is the first BTV-12 isolated from India and the second BTV-12 to be sequenced worldwide. The analysis of sequences of this virus suggests that NMO1 derived its segments from viruses belonging to western topotype viruses, as well as those from South-East Asia and India. The results have implications for understanding the origin, emergence/re-emergence and movement of BTV as well as for the development of vaccines and diagnostics based on robust epidemiological data., (© 2013 Blackwell Verlag GmbH.)

Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting., (© 2013 Blackwell Verlag GmbH.)

Bluetongue (BT) was monitored in wildlife in France during two consecutive years corresponding to contrasting incidence rates in livestock: in 2008 at the peak of domestic outbreaks and in 2009 when very few outbreaks were observed. The disease status of 2 798 ruminants comprising 837 red deer (Cervus elaphus) was explored using ELISA test on serum and real-time RT-PCR test on blood or spleen. A large proportion of red deer were seropositive and positive to RT-PCR in 2008, but also in 2009 (seroprevalence: 47.1% and 24.3%), suggesting that red deer could maintain infection when domestic incidence was negligible. By contrast, low seroprevalence (<3%) and few RT-PCR positive results were observed in other wild ruminant species, which rather appeared thus as dead-end hosts. The risk factors of bluetongue circulation during the periods of high (2008) and low (2009) domestic incidence were explored in red deer using logistic mixed models. In this species, prevalence has been mainly influenced by the initial peak of BT in livestock, but also by environmental factor such as elevation and edge density between forest and pastures. Surprisingly, cattle density has a negative influence on prevalence in red deer, possibly due to the protective effect of cattle regarding midges' bites and/or to still unexplained factors dealing with the host/midge interface. To our knowledge, this study is the first attempt at measuring the effect of landscape and wildlife/domestic interface on BT prevalence in wildlife in Europe., (© 2013 Blackwell Verlag GmbH.)

Outbreaks of bluetongue disease have occurred in Spain six times and have been caused by the following serotypes of bluetongue virus (BTV), in chronological order: BTV10, BTV2, BTV4, BTV1 and BTV8. Serotypes BTV1, BTV2 and BTV4 may have entered the country in Culicoides transported by wind; BTV8 via infected animal movements; and BTV10 across the Portuguese border. The evolution of each serotype has been different: BTV1, BTV4 and BTV10 spread throughout mainland Spain; BTV2 did not spread from the Balearic Islands to the Iberian Peninsula; and BTV8 has proven very poor at spreading throughout mainland Spain. The significant economic impact of the disease has led authorities to adopt control and eradication measures, which have evolved as new diagnostic tools and vaccines have become available. This review describes BTV infection in Spain, and it focuses on the clinical disease produced by each serotype, the Culicoides species which were present at what time, the origin of the virus and the control measures adopted. In the field, it has proven necessary to vaccinate livestock against each new BTV serotype as it arrived. Therefore, future eradication strategies should focus on developing polyvalent vaccines and vaccines that allow the differentiation of infected and vaccinated animals. As of 1 January 2013, the Iberian Peninsula is considered a restricted area for BTV1, and a small zone in southern Spain is a restricted area for BTV4, which includes the little BTV8 restricted area. Serotypes BTV1 and BTV4 were detected in sentinel animals in January and November and in March 2012, respectively. The last BTV8 positive animal was detected in November 2010, which implies that in the coming months, Spain may be declared free of BTV8., (© 2013 Blackwell Verlag GmbH.)

The recent outbreak caused by Schmallenberg virus, which affected sheep, goats and cattle in Europe, highlighted the importance of having a robust surveillance plan capable of monitoring abortions and malformations in the livestock offspring. In this context, bluetongue viruses (BTVs) represented and represent one of the major threats to the European livestock industry. Aiming to improve the understanding on BTV cross placental transmission and serotype involvement, in this retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines, 155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation. BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7-3.4%): 24 (3.6%; 95% CI: 2.4-5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6-3.0%) from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2-3.5%) from the spleen of a caprine foetus. All foetuses were from animals vaccinated with either BTV-2 or BTV-2, and BTV-9 modified live vaccines (MLVs) produced by Onderstepoort Biological Products (OBP), South Africa. Among the 31 isolated vaccine strains, serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2 (n = 3). In two cases infectious vaccine strains were found in the foetal tissues 2 months after the vaccine administration. Other pathogens known to be causative agents of abortion in ruminants were not detected nor isolated. This study demonstrates, for the first time, that BTV-2 and BTV-9 vaccine strains are able to cross the placental barrier of sheep, cattle and goats. BTV-2 and BTV-9 vaccine strains are able to infect foetuses and cause abortions or malformations depending on the period of pregnancy at the time of vaccination., (© 2012 Blackwell Verlag GmbH.)

Forty-one cattle from seven Belgian farms and two French farms confirmed as infected with bluetongue virus serotype 8 (BTV-8) were monitored from the onset of clinical signs to describe the disease pattern and estimate the duration of blood RT-qPCR and competitiveELISA positivity under field conditions. On each visit, blood samples were taken, and a standardized clinical form was filled in for each animal. A clinical score was calculated for every week until the end of clinical signs. A classification and regression tree (CART) analysis was conducted to determine the most important clinical signs every week for the first 7 weeks. The highest scores were recorded within 2 weeks of clinical onset. The first recorded clinical signs were quite obviously visible (lethargy, conjunctivitis, lesions of nasal mucosa, nasal discharge). Skin lesions, a drop in milk production and weight loss appeared later in the course of the disease. A biphasic pattern regarding nasal lesions was noticed: the first peak concerned mainly congestive and ulcerative lesions, whereas the second peak mainly concerned crusty lesions. The median time estimated by survival analysis to obtain negative RT-qPCR results from the onset of clinical signs was 195 days (range 166-213 days) in the 23 cattle included in the analysis. Serological results remained strongly positive until the end of the study. These results should ensure more accurate detection of an emerging infectious disease and are of prime importance in improving the modelling of BTV-8 persistence in Europe., (© 2012 Blackwell Verlag GmbH.)

Bluetongue virus serotype-1 (BTV-1) was isolated from Culicoides oxystoma vectors captured on livestock farms in two places of Gujarat, India. The viruses were isolated on BHK-21 cells, which produced characteristic BTV-related cytopathic effects between 24 and 48 h post-infection. Virus antigen was demonstrated in infected cells at different passage by a BTV-specific sandwich ELISA. Further, polyacrylamide gel electrophoresis and silver staining of viral genomic RNA revealed ten double-stranded RNA segments characteristic of BTV. Serotype of the isolates was identified by virus neutralization and PCR coupled with sequencing. The isolates were designated as SKN-7 and SKN-8 and their genome segment-2 (VP2) were sequenced. Phylogenetic analyses revealed very close relationship between them although they are not identical. SKN-8 showed closer relationship with a recently isolated BTV-1 from goat. Bluetongue virus was earlier isolated from Culicoides in adjacent state more than 20 years ago, although the serotype of the virus was not determined., (© 2011 Blackwell Verlag GmbH.)

After a short winter break, bluetongue virus serotype 8 was responsible in 2007 for a large-scale epidemic among ruminant populations in Western Europe. Little is known about the mechanisms allowing the virus to survive winter conditions. A yearly mass vaccination of cattle and sheep started in spring 2008, which was recognized as successful in terms of clinical protection, but occult circulation of the bluetongue virus has not been adequately addressed. We studied the carriage of bluetongue RNA in the spleen of cattle in the vector-free period and the circulation of bluetongue virus in cattle populations in Belgium since the introduction of vaccination programmes. Overall, the results presented here show evidence for the long-term carriage of bluetongue virus RNA in the spleen of cattle and demonstrated a low but significant circulation and transplacental transmission of bluetongue virus in Belgian cattle in 2009, with apparent disappearance in 2010., (© 2011 Blackwell Verlag GmbH.)

Bluetongue (BT) is an orbiviral disease of wild and domestic ruminants, mainly sheep. In Sicily, the first Bluetongue outbreak occurred in October 2000; there have been 76 recorded outbreaks so far. The National Surveillance Plan, based on European Union Commission Decision 138/2001/CE, establishes serological and entomological surveys. This plan consists of controls of seronegative cattle, called 'sentry' as indicators for the presence and circulation of virus in defined areas. To check the seroconversions, the regional territory has been subdivided in 400 km(2) areas including 58 seronegative cattle, periodically checked by serological tests. All positive sera have been tested to detect the specific serotype by the National Reference Centre for Exotic Diseases (CESME) at the Istituto Zooprofilattico Sperimentale Abruzzo e Molise in Teramo (IZS Teramo). Moreover, entomological surveillance has been implemented in seropositive herds, to investigate the presence of insect vectors belonging to Culicoides genus. The goal of the present communication is to report on the different species of Culicoides found in the farms with Bluetongue virus and to investigate on the probable role of new competent vectors. This paper concerns data analysis of 581 light-trap catches collected in 321 farms from 2003 to 2008. We observed that 82% of checked farms were positive for Culicoides spp., and only 10% of the farms were positive for Culicoides imicola.

After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

Bluetongue (BT) is an arthropod-borne viral disease of ruminants. In August 2006, domestic ruminant populations in Northern Europe became infected with BT virus serotype 8 (BTV-8). The first BTV-8-case of the year 2007 in Belgium was notified in July. This case was the starting point of a second wave of BT outbreaks. The main objective of this study was to describe the evolution and the clinical impact of the second episode of BT in Belgium. In addition, the main differences with the previous episode (August-December 2006) are reported. Both outbreak and rendering plant data were analysed. Overall cumulative incidence at herd level was estimated at 11.5 (11.2-11.8) and 7.5 (7.3-7.8) per cent in cattle and sheep populations respectively. The findings went in favour of a negative association between within-herd prevalence in 2006 and the risk of showing clinical signs of BT in 2007 (via protective immunity). A high level of correlation was demonstrated between BT incidence and small ruminant mortality data when shifting the latter of 1-week backwards. This result supports the hypothesis that the high increase in small ruminant mortality observed in 2007 was the consequence of the presence of BT. For cattle, the correlation was not as high. An increase in cattle foetal mortality was also observed during the year 2007 and a fair correlation was found between BT incidence and foetal mortality.

Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

Bluetongue virus (BTV), a member of the Orbivirus genus, has contributed to great economical losses in countries across the Mediterranean basin. Although BTV has an African origin, it has been reported in the South of Europe since 1924 when it was first detected in Cyprus. After this first Bluetongue (BT) outbreak, many others followed in most Mediterranean countries, resulting in seven BTV serotypes detected in 17 countries in Central-, South-Europe and North Africa in the last 10 years. Currently, six BTV serotypes (1, 2, 4, 8, 9 and 16) are circulating throughout Central- and Western Europe. The unexpected occurrence of BTV serotype 8 in Central Europe in 2006 and its spread and persistence have evidenced changes in the BTV scenario: new serotypes, never detected in the Mediterranean area so far, are playing a central role in the maintenance of BTV in Europe, and new species of Culicoides are now confirmed to be able to transmit the virus. Therefore, it is necessary to improve and implement specific assays in order to identify the serotypes currently present in different Mediterranean countries in a fast and reliable way. In this study, we present a new gel-based and real-time RT-PCR assay for the detection of BTV serotype 4. The sequence amplified in this test is located within BTV segment 2, a variable region of BTV dsRNA genome encoding the major outer-capsid protein VP2. No cross-reaction has been shown with other genetically or geographically related viruses and the sensitivity of this test allows the detection of 1.5-15 TCID50/ml of BTV.