Ornithodoros coriaceus (pajaroello tick) as a vector of bluetongue virus

Preliminary studies demonstrated that the argasid tick, Ornithodoros coriaceus Koch, could become infected with bluetongue virus (BTV). Ticks became infected after feeding through artificial membranes on BTV-infected suspensions of cell cultures, chicken embryos, and sheep blood. Ticks also became infected after natural feeding on viremic sheep (BTV serotype 17) and cattle (BTV serotype 11). Virus was recovered from the hemolymph and salivary glands of ticks which had ingested BTV either through an artificial membrane or by natural feeding on a host animal. Ticks infected with BTV serotype 13 were capable of transmitting the virus to a susceptible cow at 42 days after ingestion of virus-infected cultures, thus demonstrating the potential of the tick to serve as a biological vector of BTV.     

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.     

Bluetongue and epizootic hemorrhagic disease virus isolation from vertebrate and invertebrate hosts at a common geographic site

One serotype of bluetongue virus (BTV) and two serotypes of epizootic hemorrhagic disease virus (EHDV) were isolated from vertebrate and invertebrate hosts on a farm in Colorado. The isolations were from blood samples collected a week apart from a dairy heifer with stomatitis and laminitis; EHDV serotypes 1 and 2 were isolated from the first blood sample, and BTV serotype 13 and EHDV serotype 1 were isolated from the second. Antibodies to EHDV and BTV were detected in the serum from this heifer. Both EHDV serotypes and BTV serotype 13 were isolated from pools of female biting gnats (Culicoides variipennis) that had not had a recent blood meal. The BTV insect isolate was biologically transmitted by female gnats from an infected donor sheep to a recipient host sheep. Culicoides variipennis was the predominant insect collected during three nights of light trap captures at the farm.     

Genetic stability in calves of a single strain of bluetongue virus

Newborn calves were inoculated IV with highly plaque-purified bluetongue virus (BTV), serotype 10. The electrophoretic migration patterns of RNA segments and proteins of viruses isolated from calves at intervals after inoculation were compared. In addition, sera collected from calves at intervals after inoculation were compared for their abilities to neutralize several virus isolates from the same calf. Viremia persisted in calves for up to 56 days. Differences were not detected in the electrophoretic migration pattern of RNA segments or proteins of any of the BTV isolates. All calves produced high titers of neutralizing antibody to the original BTV inoculum by 28 days after inoculation, and significant (greater than or equal to 4-fold) differences were not detected in the neutralizing titers of sera to viruses collected at intervals after inoculation. The plaque-purified strain of BTV appeared to be stable genetically in infected calves, and failure to demonstrate antigenic variation among isolates indicated that antigenic shift was not the mechanism that allowed viremia to persist in BTV-infected calves.     

Epizootiologic study of bluetongue: virologic and serologic results

Heparinized blood and serum samples were obtained from 1,295 ruminants in herds or flocks with bluetongue virus (BTV) infection in 4 western states. Submissions were from herds or flocks with clinical bluetongue (BT), as well as from animals on premises with no history of BT disease. Insects, including Culicoides variipennis, were collected in areas enzootic for BT disease. Viral isolations were in 10-day-old embryonating chicken eggs that were then adapted to Vero cells for serotyping. Sera were tested from group-specific antibody to BTV by the micro agar gel precipitin (AGP) test. Viral isolations were from cattle (81), sheep (122), goats (9), antelope (2), and C varipennis (5). There were 7 isolates of serotype 120, 114 of serotype 11, 42 of serotype 13, and 56 of serotype 17. In herds or flocks from which BTV was isolated, 51% of cattle, 56% of sheep, 21% of goats, and 52% of antelope had AGP antibodies. Virus was isolated from 43% of the cattle and 23% of the sheep that had no demonstrable evidence of AGP antibodies. Viral isolations were seasonal, occurring from August until December. Approximately 30% of the herds or flocks from which virus was isolated had more than one serotype of virus causing infection.     

T lymphocyte subset alterations following bluetongue virus infection in sheep and cattle

To determine potential mechanisms of differential disease expression in ruminants infected with bluetongue virus (BTV), clinically normal, BTV-seronegative, yearling sheep and cattle were infected subcutaneously with a standardized insect-source inoculum of BTV serotype 17 (BTV-17) (three infected and one contact control each) or animal adapted BTV serotype 10 (BTV-10) (three sheep only). BTV was isolated from peripheral blood cell components of infected sheep and cattle and all infected animals showed evidence of seroconversion by 14 days post infection (PI). Sheep infected with both serotypes of BTV developed pyrexia, oral lesions, and leukopenia which were most severe on days 7-8 PI. Analysis of peripheral blood mononuclear leukocytes with specific monoclonal antibodies and flow cytometry revealed panlymphocytopenia on day 7 PI. This response was further characterized by an increase in the CD4/CD8 ratio (greater than 3) resultant from a greater decrease in absolute numbers of circulating SBU-T8(CD8+) ("cytotoxic/suppressor") lymphocytes compared to SBU-T4 (CD4)+ ("helper") lymphocytes. SBU-T19+ lymphocytes were also decreased below baseline values on days 5-14 post infection. On day 14 PI there were increased CD8+ lymphocytes and decreased CD4/CD8 ratios (approximately 0.6) in these sheep. Clinical and hematologic changes in cattle infected with BTV-17 were minimal and consisted of mild pyrexia (rectal temperature 103 degrees F) on day 9 PI in two of three infected animals and mild leukopenia on several days PI in one animal. This leukopenia was the result of a pan T lymphocytopenia with CD4/CD8 ratios in the expected range (1-2). Similar to infected sheep, infected cattle did have a shift (decrease, approximately 0.8) in the peripheral CD4/CD8 ratio associated with an increase in circulating BoT8 (CD8)+ lymphocytes on day 14 post infection. Lymphocytes in the peripheral blood of all sheep and cattle infected with BTV-17 proliferated in vitro in response to purified BTV-17. These results confirm and extend those of previous studies that indicate species differences in the hematologic response to an equivalent BTV infection in domestic ruminants.     

Susceptibility of a fetal tongue cell line derived from bighorn sheep to five serotypes of bluetongue virus and its potential for the isolation of viruses

A cell line (BHFTE) was derived from a tongue explant of a bighorn sheep fetus (Ovis canadensis nelsoni). The cells have been maintained through 23 serial passages, and the modal number of chromosomes was calculated to be 55. Monolayer cultures were shown to be susceptible to various viruses, including bluetongue virus (BTV). Of 5 BTV serotypes (2, 10, 11, 13, and 17) tested, each produced a cytopathic effect (CPE) on initial passage at 33 C. A field isolate (serotype 10) of BTV from a black-tailed deer (Odocoileus hemionus columbianus) in its second passage in Vero-M cells also produced CPE when inoculated into BHFTE cells. Antigens of BTV were demonstrated by direct immunofluorescence in the cytoplasm of BHFTE cells inoculated with homogenates of chicken embryos injected with clinical specimens from a domestic sheep and an Arabian oryx (Oryx gazella leucoryx). A suspension of BTV-infected gnats (Culicoides spp.) produced CPE and BTV-specific fluorescence on the first passage in cells inoculated with a suspension of blood from sheep experimentally infected with BTV. Additionally, selected bovine viruses induced CPE in the cells. The cell line, which is free of mycoplasma and bovine viral diarrhea virus contamination, may be useful in diagnostic medicine and research involving the ruminant species.     

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.     

Effect of Inter‐Breed Embryo Transfer on Lamb Growing Performance and Survival

The roles of recipient and embryo genotype in determining the growth performance and survivability of offspring based on specific behavioural differences were investigated using inter‐breed embryo transfer. This study was carried out using three recipient genotypes (Awassi, Redkaraman and Tuj) and two embryo genotypes (Charollais and Romanov) to obtain the six possible combinations of ewe and lamb genotypes. Data were collected from 71 recipient ewes (10 Redkaraman with Charollais and 15 Redkaraman with Romanov embryos; 10 Tuj with Charollais and 12 Tuj with Romanov embryos, and 12 Awassi with Charollais lambs and 12 Awassi with Romanov embryos); all ewes received two frozen‐thawed embryos. Awassi ewes had a significantly longer duration of the licking/grooming event (25.5 min, p < 0.05) than Tuj ewes. Charollais lambs were significantly (p < 0.05) more likely to require birth assistance compared to Romanov lambs. Romanov lambs were significantly more (p < 0.01) active than Charollais lambs in the first 2 h after birth; ewe breed had no effect on lamb behaviour. There was no recipient breed effect on either birth or weaning weights of lambs. Charollais lambs were recorded with higher birth (5.5 ± 0.3 kg vs 3.9 ± 0.2 kg; p < 0.001) and weaning (29.4 ± 1.2 kg vs 22.4 ± 1.9 kg; p < 0.001) weights compared to Romanov lambs. At weaning Romanov lambs had significantly higher (95% vs 75%; p < 0.05) survival rates, however, this was not significantly affected by recipient breed. It was concluded that recipient breed was not an important factor in survival and weaning performance of embryo transferred lambs from a prolific breed (Romanov) while these traits recorded for lambs from meat type (Charollais) embryos were influenced by dam breed.     

Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry

The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.     

Genetic variation of bluetongue virus serotype 11 isolated from host (sheep) and vector (Culicoides variipennis) at the same site

Genetic relatedness of 2 strains of bluetongue virus (BTV) serotype 11 that were isolated from the same geographic site--one from host (sheep) and the other from the vector Culicoides variipennis during an enzootic of bluetongue at Bruneau, Idaho, in August 1973--was determined by comparing the oligonucleotide fingerprint analyses of the individual double-stranded RNA segments of the genomes. It was observed that the 2 strains of BTV-11 exhibit considerable differences in their genotypes, the percentage of diversity being different for each of the corresponding RNA species of the 2 strains of BTV-11. These results indicate that more than one genotype of BTV can circulate in juxtaposition in a given geographic site. The observed genotypic diversity might be due to the accumulation of point mutations on specific RNA species or antecedent reassortment of RNA segments between different BTV in nature or both.     

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds; mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjuctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

Transplacental transmission of Bluetongue virus serotype 8 in ewes in early and mid gestation

The ability of Bluetongue virus serotype 8 (BTV-8) originating from the 2006 European outbreak to cross the ovine placenta during early and mid gestation was investigated in two separate experiments. In the first experiment, 16 ewes were infected with BTV-8 at 70-75 days gestation. The foetuses were collected at 18-19 days after infection (dpi). BTV-8 could be isolated from at least two organs of 19 out of 40 lambs and from 11 out of 16 infected ewes. In the second experiment, 20 BTV-8 infected ewes in early gestation (day 40-45) were euthanized at 10 days (10 ewes) or 30 days (10 ewes) after infection. The presence of BTV could be demonstrated in two foetuses from two ewes at 10 dpi and in 4 foetuses from four ewes at 30 dpi. The main pathological findings in the foetuses in mid gestation were meningo-encephalitis and vacuolation of the cerebrum. In the foetuses early at gestation, haemorrhages in various foetal tissues and necrosis and haemorrhages in the placentomes were found. These experiments demonstrate for the first time the presence of infectious BTV in lamb foetuses at different stages of gestation, combined with a difference in transmission rate depending on the gestation stage. The high transmission rate found at mid term gestation (69%) makes our model very suitable for further research into the mechanisms of transplacental transmission and for research into the reduction of this route of transmission through vaccination.     

Experimental transmission of bovine viral diseases by insemination with contaminated semen or during embryo transfer

Three experimental approaches were used to study transmission of blue tongue (BT), infectious bovine rhinotracheitis (IBR) and bovine virus diarrhoea (BVD) viruses. These were insemination with contaminated semen, experimental infection of embryo donor cows, or transfer of embryos experimentally exposed to virus in vitro to normal recipients. Parameters assessed included number and quality of embryos produced, virus detection (isolation and electron microscopy), serology and histopathology. All superovulated sesceptible cows inseminated with semen containing blue tongue virus (BTV) (n = 2) or infectious bovine rhinotracheitis virus (IBRV) (n = 2) became infected. One cow inseminated with semen containing BTV produced seven virus-free seven-day-old embryos; the second cow failed to produce any embryos. One of two cows inseminated with semen containing IBRV produced two underdeveloped, virus-free embryos while no embryos were produced by the second cow. One of two cows inseminated with semen containing bovine viral diarrhoea virus (BVDV) became infected. Two poorly developed, virus-free seven-day-old embryos were recovered from one of these cows. Superovulated susceptible cows inoculated either intramuscularly with BTV (n = 3) or intranasally with IBR virus (n = 2) became infected. Virus was isolated from some tissues of two BTV-infected cows, neither of which produced embryos. A third BTV-infected cow produced two virus-free embryos collected at necropsy five days after inoculation. One of two cows experimentally infected with IBR virus, produced three embryos but virus was not detected either by electron microscopy (1 embryo) or in cell culture by cytopathic alterations (1 embryo).(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of bluetongue virus in bovine fetuses using the avidin-biotin complex immunoperoxidase method

The avidin-biotin complex immunoperoxidase technique was adapted for use in detecting bluetongue virus (BTV) antigens in BTV serotype 11-infected bovine fetuses. Fetuses were infected with BTV serotype 11 at 120 days of gestation and then removed 20 days later by Cesarean section. Blood and tissue samples were collected from each animal and used for virus isolation in embryonated chicken eggs, the immunofluorescent antibody test, and the avidin-biotin complex test. The avidin-biotin complex method successfully identified BTV antigens in both fresh and autolyzed fetal brains. Thus, the avidin-biotin complex immunoperoxidase method has potential as a possible procedure for diagnosing bluetongue disease in aborted bovine fetuses.     

Maximal predicted duration of viremia in bluetongue virus-infected cattle.

Central to the development of rational trade policies pertaining to bluetongue virus (BTV) infection is determination of the risk posed by ruminants previously exposed to the virus. Precise determination of the maximal duration of infectious viremia is essential to the development of an appropriate quarantine period prior to movement of animals from BTV-endemic to BTV-free regions. The objective of this study was to predict the duration of detectable viremia in BTV-infected cattle using a probabilistic modeling analysis of existing data. Data on the duration of detectable viremia in cattle were obtained from previously published studies. Data sets were created from a large field study of naturally infected cattle in Australia and from experimental infections of cattle with Australian and US serotypes of BTV. Probability distributions were fitted to the pooled empirical data, and the 3 probability distributions that provided the best fit to the data were the gamma, Weibull, and lognormal probability distributions. These asymmetric probability distributions are often well suited for decay processes, such as the time to termination of detectable viremia. The analyses indicated a > 99% probability of detectable BTV viremia ceasing after < or = 9 weeks of infection in adult cattle and after a slightly longer interval in BTV-infected, colostrum-deprived newborn calves.     

The impact of naturally-occurring,trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the ‘over-wintering’ of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008.The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n = 4); (2) fetal malformations suggestive of BTV infection (n = 10); (3) positive PCR results from red blood cells in-lambs (n = 7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n = 9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.     

Production of ovine chimeras by inner cell mass transplantation

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.