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.     

Bluetongue in exotic sheep in Cameroon

Summary Bluetongue virus (BTV) was diagnosed in the Animal Research Institute, Mankon, Bamenda from tissue samples collected from five sheep of exotic breeds which had died of suspected bluetongue in a series of outbreaks between June and October 1982. Five serotypes BTV 1, 4, 5, 12 and 14 were isolated during the period mentioned. Similar disease occurred during June of the following year and BTV type 16 was isolated from a spleen sample from a dead sheep.

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Resumen Se diagnosticó lengua azul, mediante aislamiento del virus, en el Instituto de Investigaciones Animales, Mankon, Bamenda, de muestras de tejido colectades de cinco ovejas de razas exóticas, las cuales murieron con sospecha de lengua azul, en una seríe de brotes entre junio y octubre de 1982. Se aislaron cinco serotipos BTV 1, 4, 5, 12 y 14 durante el período mencionado. La misma enfermedad occurió en junio del a?o siguiente, aislándose BTV tipo 16 del bazo de una oveja enferma.

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Résumé A l'Institut de Recherches zootechniques de Mankon Bamenda, le virus de la fièvre catarrhale a été isolé à partir de prélèvements de tissus pris sur cinq moutons de races exotiques qui sont morts d'une maladie soup?onnée être la fièvre catarrhale lors d'une série d'épidémies de juin à octobre 1982. Cinq sérotypes (BTVI) 4, 5, 12 et 14) ont été isolés pendant la période mentionnée. Une maladie similaire est apparue pendant le mois de juin de l'année suivante et le sérotype BTV 16 a été isolé à partir d'un prélèvement de rate pris sur un mouton mort.

     

Bluetongue virus in the French Island of Reunion

This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.     

Bluetongue virus serotype 26: infection kinetics and pathogenesis in Dorset Poll sheep

Bluetongue virus serotype 26 (BTV-26) has recently been isolated from sheep in Kuwait. The aim of this study was to assess the pathogenicity and infection kinetics of BTV-26 in Dorset Poll sheep. Six sheep were experimentally infected with BTV-26 and samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and two group specific ELISAs. Five of the six sheep showed mild clinical signs characteristic of bluetongue including conjunctivitis, reddening of the mouth mucosal membranes, slight oedema of the face and nasal discharge. Viral RNA was detected in 5 of the 6 sheep by real time RT-PCR, however the levels of viral RNA detected in the samples were lower and of shorter duration than seen with other field strains of BTV. Virus was isolated from the blood of infected animals at the peak of viraemia at around 9 dpi. Antibodies against BTV were first detected by 7 dpi using the early detection BTV ELISA and a little later (7-14 dpi) using a BTV specific competitive ELISA. Four of the five remaining sheep developed neutralising antibodies to BTV-26, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 1.40 to 2.08.     

Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.     

Bluetongue virus serotypes 20 and 17 infections in sheep: Comparison of clinical and serological responses

The clinical, virological and serological responses of sheep infected with an Australian bluetongue virus (BTV) isolate (serotype 20) were compared to responses in sheep inoculated with an American bluetongue isolate (serotype 17) with which it had shown cross-reactions in serum neutralization tests. In sheep inoculated with BTV 20, clinical signs were very mild and viremia was first detected by day 5; virus was isolated intermittently for a further 2 to 3 days. Neutralizing and precipitating antibodies were first detected in the serum of the sheep between 2 to 3 weeks following inoculation. In contrast, sheep inoculated with BTV 17 showed pyrexia and severe hyperemia of the nasolabial area and oral mucosa from day 7 to 17. Viremia was first detected on day 3 and extended to day 20, while the appearance and titers of serum antibodies was similar in both groups.After challenge with BTV 17 the sheep in both groups remained clinically normal, and virus was not detected in the blood; however, serum neutralizing antibody titers to both viruses increased 2 weeks after challenge and the mean titer of the two groups ranged from 1:250 to 1:640.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Immunologic response of sheep to inactivated and virulent bluetongue virus

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.     

Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria

This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a ‘western’ BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.     

应用Nested—PCR技术检测人工感染绵羊蓝舌病病毒

实验采用6只绵羊分成3组,一组作为对照接种BHK21细胞上清液,另二组分别接种BIV10和中国分离株Z1株,攻毒定期采血提取抗原并提取病毒RNA,同时进行琼脂扩散试验、普通PCR、Nested-PCR试验。结果在整个攻毒期间,琼脂扩散方法检测不到抗原,普通PCR方法只能在攻毒后12d-15d测得抗原,而Nested-PCR在攻毒后6d即可测得病毒RNA,并且可一直持续到30d。试验证明,Nested-PCR方法是高度敏感的检测抗原的方法。在临床上能够较早地检出抗原,这对于及时采取有效措施,控制疾病流行具有积极的意义。     

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.     

光生物素cDNA探针对体内外蓝舌病病毒感染的检测

蓝舌病病毒(BTV)基因的第三片段(RMA_3)在不同型间有较高的同源性.用光生物素标记的其cDNA重组体pC7,检测2～22型BTV BHK细胞培养物全部为阳性,而相关病毒EHDV_1、EHDV_2和Ibraki病毒为阴性;同一探针检测17个型BTV攻毒羊的全血样品均为阳性,未感染的正常羊血细胞和BHK细胞培养物样品均为阴性.     

Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep

Donor sheep were infected either by bites of bluetongue virus (BTV)-infected (serotype 11, "Texas Station strain") Culicoides variipennis or by inoculation with 100,000 median chicken embryo intravascular lethal doses of BTV (serotype 11) from a suspension made from infected C variipennis. Fourteen embryos from 4 BTV-infected ewes bred by rams not infected with BTV were transferred to 8 BTV-seronegative recipient ewes, and 35 embryos and 4 unfertilized eggs from 14 BTV-infected ewes bred by BTV-infected rams were transferred to 19 BTV-seronegative recipient ewes. Eleven pregnancies and 12 lambs resulted. None of the recipients or lambs seroconverted, and BTV was not isolated from the pregnant recipient ewes or their lambs at slaughter 30 days after parturition.     

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.     

Involvement of the skin during bluetongue virus infection and replication in the ruminant host

ABSTRACT: Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence". BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/ macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.     

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.     

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.     

Clinical and serological response of sheep to serial challenge with different bluetongue virus types

A group of British sheep was infected with bluetongue virus 5 (BTV5) and subsequently challenged with the same virus type. Protection from this challenge and a homotypic BTV neutralising antibody response were observed. A second group of sheep was infected serially with three different BTV types. Animals previously exposed to BTV4 and BTV3 were found to be resistant to challenge by BTV6. Animals infected with BTV4 and challenged with BTV3 were shown to produce a transient heterotypic neutralising antibody response to a number of types. Although the level of this heterotypic response diminished with time, after challenge with BTV6 these animals developed a similar broad heterotypic response. The nature of this response and its implications in terms of observed protection merit consideration in future vaccine design and evaluation of field survey work.     

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.