Introduction: The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Three independent 1-year studies were conducted during 3 consecutive years to better define the prevalence of bluetongue virus (BTV) infection in Mexico. Serologic data were obtained by use of agar-gel immunodiffusion for identification of BTV group-reactive antibodies, and virologic data were obtained by virus isolation. Samples were obtained from sheep in 6 states over a 1-year period, with 9% seropositive; samples were obtained from cattle in 11 states during the same 1-year period, with 35% seropositive. Two years later, samples were obtained from cattle in 4 additional states, with 69% seropositive. Virus isolation was conducted on pooled blood samples obtained from cattle in 7 states. Six virus isolates were recovered and included 2 isolates each of BTV serotypes 11 and 13 and 1 isolated each of serotypes 10 and 17. All virus isolates were partially characterized by electrophoretic analysis of genomic RNA migration profiles (electropherotypes) in polyacrylamide gels. All Mexican isolates of BTV differed considerably in electropherotype profile, as compared with their respective US prototype strain of the same serotype. Such differences appeared to be much more extensive than those described to exist between numerous California isolates of the same serotype.

Bluetongue (BT) is a vector-borne disease of ruminants caused by Bluetongue virus (BTV). Twenty-nine different serotypes of BTV are currently reported throughout the world. The main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple BTV serotypes, which the conventional vaccines fail to address. To this end, three different BTV proteins (conserved region of viral protein [VP]2, VP5 and NS1) were expressed and purified in an Escherichia coli expression system. The immunogenicity of these proteins was tested in murine models using the MontanideTM ISA 201 VG adjuvant. BALB/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until Day 40 post-infection/vaccination. Protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. Neutralising antibody (Nab) titres and cross-neutralisation against a range of BTV serotypes (BTV-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. The recombinant proteins elicited higher Nab titres compared with the inactivated vaccine group, except for BTV-1, where the inactivated vaccine group elicited higher Nab titres. Additive effect of the three proteins was not observed as the Nab titres generated with a combination of conserved VP2, VP5 and NS1 was similar to those of the individual protein groups. Whilst BTV-12 could only be neutralised by serum raised against the inactivated vaccine group, BTV-5 and -24 could not be neutralised by any of the groups tested. Our cumulative data suggest that the conserved regions of VP2 (cVP2), VP5 and NS1 could play an important part in the novel vaccine design against multiple BTV serotypes. Importantly, given that VP2 was already known to elicit a serotype-specific immune response against BT, we report, for the first time, that the conserved region of VP2 has the ability to induce cross-protective immune response.

To evaluate herd-level risk factors for seropositive status of cattle to 1 or more bluetongue viruses. 110 herds of cattle in Nebraska, North Dakota, and South Dakota. Blood samples were collected before and after the vector season. Samples were tested for antibodies against bluetongue virus by use of a commercially available competitive ELISA. Factors evaluated included descriptors of geographic location and management practices. Trapping of insect vectors was conducted to evaluate vector status on a subset of 57 operations. A multivariable logistic regression model was constructed to evaluate associations. For the full data set, altitude and latitude were associated with risk of having seropositive cattle (an increase in altitude was associated with an increase in risk, and a more northerly location was associated with a decrease in risk of a premise having seropositive cattle). Import of cattle from selected states was associated with an increase in risk of having seropositive cattle. From the subset of herds with data on vector trapping, altitude and latitude were associated with risk of having seropositive cattle, similar to that for the full model. However, commingling with cattle from other herds was associated with a decrease in risk of seropositivity. Findings reported here may be useful in generating additional hypotheses regarding the ecologic characteristics of bluetongue viruses and other vector-borne diseases of livestock. Sentinel surveillance programs are useful for documenting regionalization zones for diseases, which can be beneficial when securing international markets for animals and animal products.

Inoculation of 53 ewes after 35, 45, 60, or 80 days of gestation with bluetongue virus serotypes 10, 11, 13, or 17, or with epizootic hemorrhagic disease virus serotypes 1 or 2, resulted in overt clinical disease in the 47 ewes inoculated with bluetongue virus but not in the 6 ewes inoculated with epizootic hemorrhagic disease virus. None of the lambs produced by these ewes had developmental defects or any evidence of persistence of viremia.

Results of testing of 19,731 samples from a serologic survey of cattle with bluetongue virus (BTV) infections in Australia were analyzed for association between age, species, or sex and test result. Bivariate analysis indicated that all 3 host factors were associated with test result. After adjusting for confounding caused by the location of each animal in the study (high, moderate, and low BTV prevalence regions), cattle greater than or equal to 4 years old had an odds ratio of 4.33 (95% confidence interval, 3.99, 4.71) for a positive test result, compared with that for cattle < 2 years old. Cattle 2 to 4 years had an odds ratio of 2.28 (2.14, 2.54), compared with cattle < 2 years old. Bos taurus cattle had an odds ratio of 1.76 (1.63, 2.05) of a positive test result, compared with crossbred cattle, and B indicus cattle had an odds ratio of 1.20 (1.09, 1.33), compared with crossbred cattle. Sexually intact (+) male cattle were found to have an odds ratio of 3.13 (2.66, 3.49) for a positive test result, compared with castrated male (-) cattle, and female cattle were found to have an odds ratio of 1.38 (1.29, 1.48), compared with male (-) cattle. Multivariate analysis of BTV testing results was performed, using stepwise logistic regression. The most parsimonious model selected included age, species, and sex factors, and first-order interaction terms between these factors. This model was only able to be fit to data from cattle restricted to the high (> 25%) BTV prevalence region. Odds ratios were found to increase with age for male (-) cattle of all species. Odds ratios were found to be greatest at 2 to 4 years of age for female cattle of all species and for B taurus and crossbred male (+) cattle.