Isolation and Cultivation of a New Isolate of BTV-25 and Presumptive Evidence for a Potential Persistent Infection in Healthy Goats.

Recently, several so-called "atypical" Bluetongue virus (BTV) serotypes were discovered, including BTV-25 (Toggenburg virus), in Switzerland. Most "atypical" BTV were identified in small ruminants without clinical signs. In 2018, two goats from a holding in Germany tested positive for BTV-25 genome by RT-qPCR prior to export. After experimental inoculation of the two goats with the BTV-25 positive field blood samples for generation of reference materials, viremia could be observed in one animal. For the first time, the BTV-25-related virus was isolated in cell culture from EDTA-blood and the full genome of isolate "BTV-25-GER2018" could be generated. BTV-25-GER2018 was only incompletely neutralized by ELISA-positive sera. We could monitor the BTV-25 occurrence in the respective affected goat flock of approximately 120 goats over several years. EDTA blood samples were screened with RT-qPCR using a newly developed BTV-25 specific assay. For serological surveillance, serum samples were screened using a commercial cELISA. BTV-25-GER2018 was detected over 4.5 years in the goat flock with intermittent PCR-positivity in some animals, and with or without concomitantly detected antibodies since 2015. We could demonstrate the viral persistence of BTV-25-GER2018 in goats for up to 4.5 years, and the first BTV-25 isolate is now available for further characterization.

Bluetongue in India: a systematic review and meta-analysis with emphasis on diagnosis and seroprevalence.

Bluetongue (BT) is an infectious viral disease which affects a wide range of ruminants and was first reported in India in 1964. In view of the absence of comprehensive information on the BT status in India, this study presents the seroprevalence on BT in farm animals of India based-on a systematic review and meta-analysis. A systematic review was conducted to identify the published articles (2001-2018) reporting the seroprevalence of BT in sheep, goats, cattle, buffalo, camels, and Mithun (Bos frontalis) from India. From 409 research articles, 71 fulfilled the inclusion criteria and meta-analysis for proportions was carried out targeting the eligible studies. From these, 144 strata level data were extracted with a sample size of 14048 sheep, 14696 goats, 5218 cattle, 2653 buffaloes, 2062 camels, and 222 Mithun. Overall, the analyses showed that the BT seroprevalence of 43% (95% CI: 38-49%) in goats, 39% (95% CI: 33-46%) in sheep, 38% (95% CI: 25-45%) in cattle, 34% (95% CI: 20-51%) in buffaloes, 16% (95% CI: 10-22%) in camels, and 66% (95% CI: 17-95%) in Mithun. Furthermore, the meta-regression analysis suggested that serological tests, geographical region, and sample size were the prime moderators. Meta-analytic study indicates the BT seropositivity in 25.35 million sheep (95% CI: 21.5-29.9), 58 million goats (95% CI: 51.3-66.2), 66.8 million cattle (95% CI: 47.7-86), 37.0 million buffaloes (95% CI: 21.7-55.4), 0.06 million camels (95% CI: 0.04-0.09), and 0.19 million Mithun (95% CI: 0.05-0.28). The findings highlight the variation of BT seropositivity in different geographical regions of India.

Method validation, reference values, and characterization of acute-phase protein responses to experimentally induced inflammation and bluetongue virus infection in the Iberian ibex.

BACKGROUND: Acute phase protein (APP) concentrations can change due to inflammation and be used to monitor disease in the Iberian ibex (Capra pyrenaica). OBJECTIVES: This study aimed to validate Haptoglobin (Hp) and serum amyloid A (SAA) analytes, establish reference values, and characterize Hp and SAA responses in the Iberian ibex after experimentally induced inflammation and experimental bluetongue virus (BTV) infection. METHODS: Sera from 40 free-ranging box-trapped ibexes were used to establish Hp and SAA reference values. Six healthy ibexes were subcutaneously injected with 5 mL of turpentine, then, blood samples were taken, and clinical evaluations were performed on days 0, 1, 2, 3, 4, 7, and 14 postinjection. Another seven ibexes were challenged with BTV. Serum Hp and SAA concentrations were quantified using commercial assays following the manufacturer's instructions. RESULTS: Intra-assay precision and linearity were acceptable for both Hp and SAA. Intra-assay variation for high and low concentration of Hp and SAA were 9.74% and 17.31% and 16.49% and 12.89%, respectively. Inter-assay variation was higher for the low APP concentrations. Reference values for the healthy Iberian ibexes were (median, minimum, and maximum values) 0.2 (0.12-0.64) g/L for Hp and 4.74 (0.05-29.54) mg/L for SAA. Both Hp and SAA acted as a moderate and a major APP, respectively, and each could distinguish animals with turpentine-induced inflammation from those without. Hp and SAA did not change in asymptomatic BTV-infected animals. CONCLUSION: This study validated Hp and SAA analytes and provided basal reference values for these analytes in the Iberian ibex. Both APPs were able to discriminate between healthy and diseased Iberian ibexes animals during turpentine-induced inflammatory processes.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

Evaluating the most appropriate pooling ratio for EDTA blood samples to detect Bluetongue virus using real-time RT-PCR.

The control of Bluetongue virus (BTV) presents a significant challenge to European Union (EU) member states as trade restrictions are placed on animals imported from BTV-affected countries. BTV surveillance programs are costly to maintain, thus, pooling of EDTA blood samples is used to reduce costs and increase throughput. We investigated different pooling ratios (1:2, 1:5, 1:10 and 1:20) for EDTA blood samples to detect a single BTV positive animal. A published real-time RT-PCR assay (Hofmann et al., 2008) and a commercial assay (ThermoFisher VetMax™ BTV NS3 kit) were used to analyse BTV RNA extracted from pooled EDTA blood samples. The detection rate was low for the onset of infection sample (0-2 days post infection (dpi); CT 36) irrespective of the pooling ratio. Both assays could reliably detect a single BTV-positive animal at early viraemia (3-6 dpi; CT 33) when pooled, however, detection rate diminished with increasing pooling ratio. A statistical model indicated that pooling samples up to 1:20, is suitable to detect a single BTV positive animal at peak viraemia (7-12 dpi) or late infection (13-30 dpi) with a probability of detection of >80% and >94% using the Hofmann et al. (2008) and VetMAX assays, respectively. Using the assays highlighted in our study, pooling at ratios of 1:20 would be technically suitable in BTV-endemic countries for surveillance purposes. As peak viraemia occurs between 7-12 days post infection, a 1:10 pooling ratio is appropriate for post-import testing when animals are sampled within a similar time frame post-import.

Prevalence and Risk Factors of Brucellosis, Chlamydiosis, and Bluetongue Among Sika Deer in Jilin Province in China.

Brucellosis and chlamydiosis are important zoonotic diseases and bluetongue virus (BTV) is an arthropod-borne viral disease of ruminants. They are widely distributed around the world, cause large economic losses, and significant harmful effects on humans. However, epidemiological information relating to transmission from commercial sika deer in China is limited. Therefore, from 2016 to 2017, 458 sika deer blood samples were collected from three cities in Jilin Province in China. The Brucella antigen and specific antibodies to Chlamydia and BTV were examined using RT-PCR, indirect hemagglutination assay, and ELISA, respectively. The prevalence of Brucella was found to be 12.9% (59/458) and the seroprevalence of Chlamydia and BTV was 14.4% (66/458) and 17.0% (78/458), respectively. Seasonality was considered a risk factor for the presence of Brucella or BTV in sika deer and the region was considered a risk factor for Chlamydia infection. These data provides reference values for both further research and disease control.

Bluetongue disease in small ruminants in south western Ethiopia: cross-sectional sero-epidemiological study.

OBJECTIVE: The status of bluetongue disease, vectors for transmission of the disease and the serotypes involved are not clearly known in Ethiopia. This sero-epidemiological study was conducted to determine the seroprevalence and associated risk factors of bluetongue in small ruminants of South Western Ethiopia. RESULT: 422 serum samples were screened for the presence of bluetongue virus (BTV) specific antibodies using competitive enzyme-linked immunosorbent assay (c-ELISA) and 30.6% (129/422) (confidence interval CI 26.2-35%) of the sheep and goat serum samples were found positive. Multivariate analysis of several risk factors like age, sex, altitude, body condition and species of animals were studied and it was observed that species of animals, age and altitude had significant influence (P < 0.05) on seropositivity to BTV. Goats showed more seropositivity to bluetongue as compared to sheep [AOR = 2.4, 95% CI (1.5-3.9), P = 0.001], adult animals were more seropositive [AOR = 3.1, 95% CI (1.9-5.1), P = 0.001] than other age groups and animals at the lowland [AOR = 3.1, 95% CI (1.5-6.4), P = 0.002] showed more seropositivity to bluetongue than midland and high land. Sex and body condition of the animals had no statistically significant (P > 0.05) effect on seropositivity to bluetongue.

Persistence of Bluetongue virus serotype 1 virulence in sheep blood refrigerated for 10 years.

This paper reports that Bluetongue virus serotype 1 (BTV-1) infected blood collected during the 2006 Sardinia (Italy) epidemic from a ewe with clinical disease and stored at ~ 5°C for 10 years, caused Bluetongue (BT)-like clinical disease and death when inoculated into a susceptible Sarda breed ram. Anatomo-histopathological examination and Real-Time Reverse Transcriptase PCR (Real-Time RT-PCR) confirmed the presence of BTV-1 in several tissues proving that the BTV-1 2006 isolate has maintained its infectivity and virulence.

Seroprevalence and Risk Factors of Bluetongue Virus Infection in Tibetan Sheep and Yaks in Tibetan Plateau, China.

Bluetongue (BT), caused by bluetongue virus (BTV), is an arthropod-borne viral disease in ruminants. However, information about BTV infection in yaks in China is limited. Moreover, no such data concerning BTV in Tibetan sheep is available. Therefore, 3771 serum samples were collected from 2187 Tibetan sheep and 1584 yaks between April 2013 and March 2014 from Tibetan Plateau, western China, and tested for BTV antibodies using a commercially available ELISA kit. The overall seroprevalence of BTV was 17.34% (654/3771), with 20.3% (443/2187) in Tibetan sheep and 13.3% (211/1584) in yaks. In the Tibetan sheep group, the seroprevalence of BTV in Luqu, Maqu, Tianzhu, and Nyingchi Prefecture was 20.3%, 20.8%, 20.5%, and 19.1%, respectively. The seroprevalence of BTV in different season groups varied from 16.5% to 23.4%. In the yak group, BTV seroprevalence was 12.6%, 15.5%, and 11.0% in Tianzhu, Maqu, and Luqu counties, respectively. The seroprevalence in different seasons was 12.6%, 15.5%, 15.4%, and 9.0% in spring, summer, autumn, and winter, respectively. The season was the major risk factor concerning BTV infection in yaks (P < 0.05). The date of the BTV seroprevalence in Tibetan sheep and yaks provides baseline information for controlling BT in ruminants in western China.

Meteorological conditions and land cover as predictors for the prevalence of Bluetongue virus in the Inner Mongolia Autonomous Region of Mainland China.

Bluetongue is a major disease of economic importance that affects ruminants worldwide. It is transmitted by species of Culicoides midges (Diptera: Ceratopogonidae). The Inner Mongolia Autonomous Region is one of the main pastoral areas for farmed sheep in Mainland China and, because of its large area, represents an ideal candidate region for the study of Bluetongue virus (BTV) distribution and prevalence characteristics. The present study conducted a detailed investigation into the spatial patterns of BTV transmission in sheep in the Inner Mongolia Autonomous Region, and assessed the inter-relationships between meteorological factors, land cover and the transmission of the virus was conducted. Reverse-transcriptase polymerase chain reaction (RT-PCR) was used for the determination of BTV infection in the surveyed animals. Between June 2013 and February 2015, 6199 sheep were subjected to virus detection and 2199 sheep (35.47%) were determined to be positive for BTV. Subsequently, a maximum entropy model (MaxEnt) was used to investigate the relationship between land cover, meteorological factors and the prevalence of BTV infection. Jackknife analysis revealed that the mean monthly temperature, rainfall and average wind speed were associated with the occurrence of BTV infection and that BTV infection positivity was significantly higher among animals from districts with a high percentage of grassland and forest area. Our findings indicate that meteorological factors and land cover may be important variables affecting transmission of BTV and should be taken into account in the development of future surveillance programmes for BTV.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody.

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Sero-epidemiology and molecular detection of Bluetongue virus in Indian ruminants.

Bluetongue (BT) is a non­contagious arthropod­borne viral disease of domestic and wild ruminants. It is endemic to India and clinical outbreaks of disease have been reported mainly in sheep, although BT is often asymptomatic in other ruminant species. In the present serological survey, a total of 576 serum samples, comprising of 416 cattle and 160 sheep, covering different agro­climatic zones of Rajasthan, Uttar Pradesh, and Karnataka states, were screened for the presence of Bluetongue virus (BTV) specific antibodies using competitive enzyme­linked immunosorbent assay (c­ELISA). Overall 73.08% (304/416) of the cattle and 53.30% (87/160) of the sheep serum samples were positive for BTV antibodies. The prevalence of BTV antibodies in cattle in different agro­climatic zones ranged between 60­80% in Rajasthan and 66­70% in Uttar Pradesh. During the study, a nested polymerase chain reaction (PCR) based on the BTV NS1 gene (genome segment 5) was optimized for detection of BTV's nucleic acid from a cell adapted strain of BTV­23, and field derived clinical blood samples. In the present study, 19/70 of cattle and 9/30 of sheep blood samples tested positive for BTV RNA by the nested PCR, which amplified specific products of 274 bp and 101 bp sizes, respectively. From this study, it can be concluded that cattle showed higher percentage of sero­positivity in comparison to sheep. The improved sero­surveillance system for BTV in endemic areas will be of great help to understand the epidemiology of BTV and to formulate effective control and preventive strategies.

Detection of antibodies against Bluetongue virus among domestic ruminants in the highlands of Nepal.

Bluetongue (BT) is one of the most economically important transboundary animal diseases. In recent years, it has been considered a disease related to climate change. A study was undertaken in 2013 in Nepal to measure the prevalence of Bluetongue virus (BTV) infection among domestic ruminants inhabiting the 3 agro-climatic zones with altitudes ranging from 150 to 2,400 metres above sea level. Twelve clusters representing the 3 altitudes were selected. The presence of antibodies against BTV was demonstrated in serum samples of sheep, goats, cattle, buffaloes, yaks/chauries, and chyangra goats (Himalayan goat) of Nepal. For this purpose, a total of 2,084 sera were collected from a population of 202 sheep, 739 goats, 590 cattle, 379 buffaloes, 105 yaks/chauries, and 69 chyangra goats between February 2013 and January 2014. The presence of antibodies against BTV was investigated using competitive enzyme-linked immunosorbent assay (c-ELISA). Of the 2,084 collected sera, 45.20% were positive for BTV antibodies. Species-wise prevalence was 17.82%, 47.50%, 53.05%, 58.05%, 7.62%, and 20.29% in sheep, goats, cattle, buffaloes, yak, and chyangra goats, respectively. Contrary to the general belief, maximum numbers of seropositive cases were recorded in buffaloes followed by cattle, goats, chyangra goats, sheep, and yak/chauries. The samples collected in the post-monsoon period (July-August is the monsoon period) show a seroprevalence higher than the pre-monsoon samples. This study shows the seroprevalence of BT in domestic ruminant population of Nepal at all altitudes. The highest prevalence has been reported in the plains of Terai followed by gradual decline in the mid-hills, and in the high mountains. Furthermore, detection of antibodies against BTV in both small and large ruminants (chyangra goats and yak/chauries) dwelling in high altitudes in the absence of BT vaccination is suggesting vector movement to the highlands as a consequence of warmer climate. These findings suggest that the climatic conditions, even at the higher elevation, are suitable for the survival of biting midges responsible for the transmission of BTV.

Development of a novel protein chip for the detection of bluetongue virus in China.

Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.

Factors Affecting Seroconversion Rates in Cattle Vaccinated with Two Commercial Inactivated BTV-8 Vaccines Under Field Conditions.

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.

Culicoides vector species on three South American camelid farms seropositive for bluetongue virus serotype 8 in Germany 2008/2009.

Palearctic species of Culicoides (Diptera, Ceratopogonidae), in particular of the Obsoletus and Pulicaris complexes, were identified as putative vectors of bluetongue virus serotype 8 (BTV-8) on ruminant farms during the epizootic in Germany from 2006 to 2009. BTV may cause severe morbidity and mortality in ruminants and sporadically in South American camelids (SAC). However, the fauna of Culicoides spp. on SAC farms has not been investigated. Therefore, the ceratopogonid fauna was monitored on three farms with BTV-seropositive SAC in Germany. Black-light traps were set up on pastures and in stables from summer 2008 to autumn 2009. Additionally, ceratopogonids were caught in emergence traps mounted on llama dung and dung-free pasture from spring to autumn 2009. After morphological identification, selected Culicoides samples were analysed for BTV-RNA by real-time RT-PCR. The effects of the variables 'location', 'temperature' and 'humidity' on the number of Culicoides caught in black-light traps were modelled using multivariable Poisson regression. In total, 26 species of Culicoides and six other genera of biting midges were identified. The most abundant Culicoides spp. collected both outdoors and indoors with black-light traps belonged to the Obsoletus (77.4%) and Pulicaris (16.0%) complexes. The number of Culicoides peaked in summer, while no biting midges were caught during the winter months. Daily collections of Culicoides were mainly influenced by the location and depended on the interaction of temperature and humidity. In the emergence traps, species of the Obsoletus complex predominated the collections. In summary, the absence of BTV-RNA in any of the analysed Culicoides midges and in the BTV-seropositive SAC on the three farms together with the differences in the pathogenesis of BTV-8 in SAC compared to ruminants suggests a negligible role of SAC in the spread of the virus. Although SAC farms may provide similar suitable habitats for putative Culicoides vectors than ruminant farms, the results suggest that geographic and meteorological factors had a stronger influence on Culicoides abundance than the animal species.

Seroprevalence of Bluetongue virus in domestic yaks (Bos grunniens) in Tibetan regions of China based on circulating antibodies.

The bluetongue (BT) is an infectious, non-contagious disease of ruminants with a substantial impact on income and welfare of animals. To date, scarce information about this disease in domestic yaks is available in Tibetan area of China. Seroprevalence of circulating antibodies to Bluetongue virus (BTV) in yaks from three regions of Tibet and Hongyuan area of Sichuan Province in China was investigated by a commercial competitive enzyme-linked immunosorbent assay kits. A total of 736 blood samples were collected during the year 2012 and 2013, and nearly 2% serum samples were found positive for BTV antibodies in Hongyuan area during the year 2012, and 4.89 and 3.88% of samples showed positive results for BTV in Tibetan and Hongyuan area, respectively, in 2013. The results indicated the occurrence of BT infection in Chinese yaks for the first time in Tibet and Hongyuan area of Sichuan Province with the presence of BTV antibodies in these ruminants.

Bluetongue disease and seroprevalence in South American camelids from the northwestern region of the United States.

In late summer/early fall of 2013, 2 South American camelids from central Washington were diagnosed with fatal bluetongue viral disease, an event which is rarely reported. A 9-year-old intact male llama (Lama glama), with a 1-day history of anorexia, recumbency, and dyspnea before death. Abundant foam discharged from the mouth and nostrils, and the lungs were severely edematous on postmortem examination. Histologically, there was abundant intra-alveolar edema with fibrin. Hemorrhage and edema disrupted several other organs. Bluetongue viral RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and serotype 11 was identified by sequencing a segment of the VP2 outer capsid gene. Approximately 1 month later, at a site 150 miles north of the index case, a 2-year-old female alpaca with similar, acutely progressive clinical signs was reported. A postmortem examination was performed, and histologic lesions from the alpaca were similar to those of the llama, and again serotype 11 was detected by PCR. The occurrence of bluetongue viral infection and disease is described in the context of seasonal Bluetongue virus activity within the northwestern United States and southwestern Canada.

Cross-sectional serosurvey and associated factors of bluetongue virus antibodies presence in small ruminants of Nepal.

BACKGROUND: Bluetongue (BT) is an infectious, insect-borne viral disease primarily affecting sheep and occasionally cattle and goats. In Nepal, BT is an emerging disease of economic importance. The objective of this study was to estimate the seroprevalence of BT virus (BTV) in small ruminants of two eco-zones of Nepal, Hills and Terai, and to identify the factors associated with virus exposure. We conducted a cross-sectional serosurvey from March 2012 through February 2013 by sampling 318 small ruminants (184 sheep and 134 goats) from seven clusters (villages) of selected vulnerable communities of Chitwan (Terai) and Lamjung (Hills) Districts of Nepal. RESULTS: Of the 318 serum sample tested, 27.9% [95% confidence interval (CI): 23.1- 33.2] were positive for BTV antibodies (25.0% sheep and 31.3% goats). Bivariate analysis indicated a positive association between seroconversion to BTV and flock size, eco-zone, breed, and contact history with cattle. Additionally, in female sheep and goats, a history of abortion was positively associated with seropositivity to BTV. However, the final multivariable model, after controlling for clustering of animals within the villages, identified only history of abortion and breed as the factors significantly associated with BT seropositivity in female sheep and goats. Based on this model, female small ruminants having a history of abortion were more likely to be seropositive compared to those without such history [Odds Ratio (OR) = 46.14 (95% CI: 11.66- 182.5)]. Exotic breeds were more likely to be seropositive compared to indigenous breeds [OR = 9.04 (95% CI: 3.08- 24.46)] while the risk for BTV seropositivity was not significantly different between indigenous and cross breeds. CONCLUSIONS: Our results showed that nearly a quarter of small ruminants in two regions of Nepal were seropositive for BTV, indicating wide exposure of small ruminants to this pathogen. We identified history of abortion and breed as factors significantly associated with the seropositivity of BTV. We recommend that surveillance for BTV infection in Nepal be strengthened and that it would be valuable to enhance the education of farmers about the possible impacts of this disease.

Evidence for transmission of bluetongue virus serotype 26 through direct contact.

The aim of this study was to assess the mechanisms of transmission of bluetongue virus serotype 26 (BTV-26) in goats. A previous study, which investigated the pathogenicity and infection kinetics of BTV-26 in goats, unexpectedly revealed that one control goat may have been infected through a direct contact transmission route. To investigate the transmission mechanisms of BTV-26 in more detail an experimental infection study was carried out in which three goats were infected with BTV-26, three goats were kept uninfected, but were housed in direct contact with the infected goats, and an additional four goats were kept in indirect contact separated from infected goats by metal gates. This barrier allowed the goats to have occasional face-to-face contact in the same airspace, but feeding, watering, sampling and environmental cleaning was carried out separately. The three experimentally infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from their blood. At 21 dpi viral RNA was detected in, and virus was isolated from the blood of the three direct contact goats, which also seroconverted. The four indirect barrier contact goats remained uninfected throughout the duration of the experiment. In order to assess replication in a laboratory model species of Culicoides biting midge, more than 300 Culicoides sonorensis were fed a BTV-26 spiked blood meal and incubated for 7 days. The dissemination of BTV-26 in individual C. sonorensis was inferred from the quantity of virus RNA and indicated that none of the insects processed at day 7 possessed transmissible infections. This study shows that BTV-26 is easily transmitted through direct contact transmission between goats, and the strain does not seem to replicate in C. sonorensis midges using standard incubation conditions.