EPIZOOTIOLOGY OF BLUETONGUE: THE SITUATION IN THE UNITED STATES OF AMERICA

Bluetongue was first reported in the United States in 1948 in sheep in Texas. The virus has now been isolated from sheep in 19 States. When the disease first occurs in a flock, the morbidity may reach 50 to 75% and mortality 20 to 50%. In subsequent years, the morbidity may be only 1 to 2% with very few deaths. Difference in breed susceptibility has not been observed. Natural bluetongue infection has not been observed in Angora or dairy goats. Bluetongue virus was first isolated from cattle, in Oregon, in 1959. The virus has now been isolated from cattle in 13 States. In cattle, the disease is usually inapparent but can cause mild to severe clinical disease and neonatal losses. Natural clinical bluetongue has also been reported in bighorn sheep, exotic ruminants in a zoo, mule deer, and white-tailed deer. Serological evidence of exposure to the virus has also been found in other species of ruminants in the wild. Inoculation of virulent bluetongue virus, vaccine virus, or natural disease can cause congenital deformities and neonatal losses in calves, lambs, and white-tailed deer fawns. Culicoides is considered the important insect vector of bluetongue. The virus has also been isolated from sheep keds and cattle lice. U.S. field strains of the virus fit into four serologic groups. No cross reactions were found between bluetongue and epizootic haemorrhagic disease of deer viruses. Cattle are considered significant virus reservoirs. It is necessary to use washed erythrocytes, rather than whole blood, and to inoculate susceptible sheep, rather than embryonated chicken eggs, to detect longer-term viraemia in cattle.     

Bluetongue and related viruses in Nigeria: Experimental infection of West African dwarf sheep with Nigeria strains of the viruses of epizootic haemorrhagic disease of deer and bluetongue

West African dwarf sheep were inoculated by the subcutaneous route with epizootic haemorrhagic disease of deer (EHD) virus or bluetongue (BT) virus. No clinical disease was observed following primary EHD or BT infection, or subsequent challenge with either homologous or heterologous virus. However, viraemia was detected in non-immune sheep exposed to BT virus, but not in BT- or EHD-immune sheep challenged with either virus, or in non-immune sheep exposed to primary EHD virus infection. Complement fixing antibodies developed against both EHD and BT viruses following the primary infection with either virus, or subsequent challenge with homologous or heterologous virus. Following a primary infection, virus-neutralising (VN) antibodies developed only against the inoculated virus, while the detection of VN antibodied to both viruses followed the challenge of an EHD- or BT-immuned sheep with either the homologous or heterologous virus. These findings further support previous reports of a relationship between EHD and BT viruses. between EHD and BT viruses.     

Studies on the Pathogenesis of Experimental Epizootic Hemorrhagic Disease of White-tailed Deer

Observations were made of clinical signs, gross and microscopic lesions in white-tailed deer infected with epizootic hemorrhagic disease (EHD) virus. Typically, animals became weak and lethargic and developed hyperemia of the oral and nasal mucosa, tongue, ears and sclera of the eyes six to seven days following intramuscular inoculation with virus. Body temperature increased initially and then fell to subnormal levels just prior to death.

A decrease in levels of circulating blood platelets was correlated with the occurrence of fever and the appearance of platelet and fibrin thrombi in small vessels of many organs of the body. Thrombosis resulted in tissue degeneration, necrosis and hemorrhages in the terminal stages of the disease. Tissues most seriously affected were oral, nasal and tongue mucosa, mandibular salivary glands, myocardium and epithelium of the forestomachs. The lesions resembled those of blue-tongue in deer.

Inoculation of domestic sheep with EHD virus-infected deer spleen tissues was without clinical effect. Blood collected from the sheep, representing the third blind passage of EHD virus in sheep, was not infective for deer.

     

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep

Objective To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.
Design A clinical and pathological study.
Procedure Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies.
Results All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection.
Conclusions The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.     

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.     

Identification of seven serotypes of bluetongue virus from the People's Republic of China

Seven serotypes (1, 2, 3, 4, 12, 15 and 16) of bluetongue virus were isolated from the blood of sheep and cattle in the People's Republic of China between 1986 and 1996. Six of these viruses were isolated in Yunnan province. The sheep from which serotypes 1 and 16 were isolated showed obvious signs of bluetongue disease, whereas the cattle from which serotypes 2, 3, 4, 12 and 15 were isolated were clinically normal. Phylogenetic analyses of these viruses indicate that they are more closely related to one another, and to an Australian strain of serotype 1, than they are to prototype strains of bluetongue virus serotypes 2, 10, 11, 13 and 17 from the USA.     

Experimentally induced bluetongue virus infection in white-tailed deer: coagulation, clinical pathologic, and gross pathologic changes

Ten yearling white-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17. Two yearling white-tailed deer were inoculated with sonicated heparinized noninfected blood and served as controls. Clinical signs of bluetongue virus infection included increased rectal temperature, erythema, facial edema, coronitis, and stomatitis. By postinoculation day (PID) 8, excessive bleeding and hematoma formation at venipuncture sites, dehydration, and diarrhea developed. At necropsy, the most consistent findings were oral lesions and widespread hemorrhage, which ranged from petechia to massive hematoma formation. Bluetongue virus caused progressive prolongation of activated partial thromboplastin time and prothrombin time, and progressive reduction of Factors VIII and XII plasma activities beginning on PID 6. A progressive decrease in platelet numbers also developed on PID 6. Changes in platelet size were not detected. Mean thrombin time was shortened, but prolongation developed in 1 deer. Mean fibrinogen concentration and Factor V plasma activity initially increased and then decreased, but remained above preinoculation values. Factor V activity was low in a few deer. Results of screening tests for inhibitors of the intrinsic coagulation system were positive in 2 deer. High concentrations of fibrin(ogen) degradation products were first detected between PID 3 and 6. Hematologic changes included leukopenia, lymphopenia, neutrophilia, and low total plasma protein concentration. Differences in PCV, hemoglobin concentration, or RBC counts were not detected between infected and control deer. Serum total bilirubin concentration increased by PID 6, primarily because of increased unconjugated bilirubin concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatine kinase activity. Indirect Coombs test results were negative in all deer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toggenburg Orbivirus, a new bluetongue virus: Initial detection, first observations in field and experimental infection of goats and sheep

A novel bluetongue virus termed “Toggenburg Orbivirus” (TOV) was detected in two Swiss goat flocks. This orbivirus was characterized by sequencing of 7 of its 10 viral genome segments. The sequencing data revealed that this virus is likely to represent a new serotype of bluetongue virus [Hofmann, M.A., Renzullo, S., Mader, M., Chaignat, V., Worwa, G., Thuer, B., 2008b. Genetic characterization of Toggenburg Orbivirus (TOV) as a tentative 25th serotype of bluetongue virus, detected in goats from Switzerland. Emerg. Infect. Dis. 14, 1855–1861].In the field, no clinical signs were observed in TOV-infected adult goats; however, several stillborn and weak born kids were reported. Although born during a period of extremely low vector activity, one of these kids was found to be antibody and viral genome positive and died 3.5 weeks postpartum.Experimental infection of goats and sheep, using TOV-positive field blood samples, was performed to assess the pathogenicity of this virus.Goats did not show any clinical or pathological signs, whereas in sheep mild bluetongue-like clinical signs were observed. Necropsy of sheep demonstrated bluetongue-typical hemorrhages in the wall of the pulmonary artery. Viral RNA was detected in organs, e.g. spleen, palatine tonsils, lung and several lymph nodes of three experimentally infected animals.Unlike other bluetongue virus serotypes, it was not possible to propagate the virus, either from naturally or experimentally infected animals in any of the tested mammalian or insect cell lines or in embryonated chicken eggs.In small ruminants, TOV leads to mild bluetongue-like symptoms. Further investigations about prevalence of this virus are needed to increase the knowledge on its epidemiology.     

Possible introduction of epizootic hemorrhagic disease of deer virus (serotype 2) and bluetongue virus (serotype 11) into British Columbia in 1987 and 1988 by infected Culicoides carried on the wind.

Outbreaks of epizootic hemorrhagic disease of deer and of bluetongue began in British Columbia in August and October 1987 respectively and recrudescence of infection by both viruses was detected the following year in August. Weather records for up to 18 days before the initial outbreaks of disease, isolation of virus or seroconversion were examined to determine if the viruses could have been introduced by infected Culicoides carried on the wind. Data on temperature, rainfall, wind speed and direction and pressure together with backward trajectory analysis showed that there were suitable winds which could have introduced Culicoides infected with epizootic hemorrhagic disease of deer virus on 13 August 1987 (14 days before disease was observed), Culicoides infected with bluetongue virus on 1 October 1987 (7 days before virus was isolated and 13 days before disease in sheep) and Culicoides infected with bluetongue or epizootic hemorrhagic disease of deer viruses on 20 July 1988 (15 days before seroconversion was detected). The arrival on 13 August 1987 coincided with the passage of a cold front and rain and that on 1 October 1987 with a fall in temperature and calm winds. The source of the Culicoides before arrival could have been the Okanogan Valley as far south as the junction of the Okanogan and Columbia rivers in Washington, USA. Flight would have been at temperatures of 12.6 degrees C or higher and at heights up to 1.5 km.     

Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

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.     

Prevalence of bluetongue virus antibodies in sheep in central Ethiopia

Competitive ELISA was applied to detect antibodies against bluetongue virus in sheep sera collected from different agro-climatic areas in Ethiopia. A total of 90 serum samples were tested and 42 (46.67%) were positive for bluetongue virus antibodies. A prevalence rate ranging from 9.67% for sheep sampled in the highland to 92.85% for sheep sampled in the lowland was recorded. The prevalence correlated with the probable distribution of the Culicoides vector. This is the first report indicating the presence of bluetongue virus infection in animals from Ethiopia.     

Epizootic hemorrhagic disease virus in Montana: isolation and serologic survey

Epizootic hemorrhagic disease virus (EHDV) was isolated in Vero cell culture from the spleen and whole blood of a white-tailed deer (Odocoileus virginianus). A 10% spleen suspension caused acute hemorrhagic disease (HD) when inoculated into an experimental white-tailed deer and resulted in the recovery of EHDV from the blood of the experimental animal at 5 days after inoculation. The virus was identified as EHDV serotype 2 through indirect fluorescent antibody tests, electron microscopy, and reciprocal cross-neutralization tests. Approximately 73% (36/49) of the mule deer, 5% (2/42) of the white-tailed deer, and 79% (249/314) of the cattle samples tested from areas where HD had been reported were EHDV seropositive. Although none of the white-tailed deer was bluetongue virus seropositive, 29% of the mule deer and 3% of the cattle tested from "active" HD areas possessed bluetongue virus precipitating antibody.     

Isolation of a virus serologically related to the bluetongue group from an outbreak of haemorrhagic disease among exotic deer in Saudi Arabia.

In February 1991 a severe haemorrhagic disease affected exotic deer aged over six months in the Al-Hofuf area of the eastern region of Saudi Arabia. The morbidity rate was 40 per cent and the case fatality rate was 60 per cent. The clinical signs were high temperature (up to 41.5 degrees C), nasal discharge, slight salivation and lacrimation, congestion of the conjunctivae, torticollis, tremors when trying to stand, recumbency and coma leading to death. Post mortem examination revealed a severe haemorrhagic disease. A virus, serologically related to the bluetongue serogroup, was isolated from the deer. Sheep and goats kept on the same farm did not show any clinical signs. The epidemiology of the outbreak is discussed.     

Serial inoculation of sheep with two bluetongue virus types

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.     

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.     

Study on the pathogenesis of bluetongue: replication of the virus in the organs of infected sheep.

The pathogenesis of bluetongue infection was studied by the titration of the virus in tissue samples taken from sheep inoculated subcutaneously in the auricula of the ear with 76 TC ID50 of the plaque-purified type 10 bluetongue virus. Tissue samples were taken from individual animals killed at daily intervals over a period of 11 days. The mean incubation time was 6.9 days and the first clinical sign was pyrexia. On the 4th day, bluetongue virus was demonstrated in the lymph nodes of the cephalic area, tonsils and spleen; viraemia became demonstrable on the 6th day post-inoculation and typical macroscopic lesions due to the virus were first observed on the 8th day. It was concluded that, post-infection, the virus entered the regional lymph nodes. From there it was disseminated via the lymph and/or the blood stream to the lymphoid tissues in other parts of the body where further replication occurred. From these primary sites the virus was carried via the blood stream and infected the majority of tissues. Humoral antibody, as detected by immunofluorescence, did not appear to have a direct influence on the concentration of virus in solid tissues. Persistence of the virus in infected sheep was not demonstrated when tissues were taken 6, 8 and 16 weeks after infection.     

Bluetongue and epizootic hemorrhagic disease in white-tailed deer from Oklahoma: serologic evaluation and virus isolation

Blood samples were collected from 194 white-tailed deer from 27 locations in Oklahoma from 1977 through 1984. Sixty-eight (35%) of the deer had antibody against bluetongue virus (BTV) and 78 (40%) had antibody against epizootic hemorrhagic disease virus. Seropositive deer were detected in each of the 4 geographic quadrants of the state. Virus isolation was attempted in 40 deer from the northeast quadrant of Oklahoma (1983 through 1984); BTV was isolated from 11 deer, but epizootic hemorrhagic disease virus was not isolated. The isolation of BTV serotype 11 from these deer from 1983 through 1984 coincided with reported isolations of this serotype in other ruminants in Oklahoma during this time.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.     

Experimental transmission of sheep-associated malignant catarrhal fever from sheep to Japanese deer (Cervus nippon) and cattle

The assumption that sheep carry ovine herpesvirus-2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), is widely accepted, albeit OvHV-2 has not been isolated. We attempted experimental contact transmission of MCF from Japanese sheep persistently infected with OvHV-2 to Japanese deer (Cervus nippon) and cattle. In Experiment 1, a deer was kept in close quarters with an infected ewe. In Experiment 2, a second deer was kept with the same ewe. In Experiment 3, two cows were each kept with two infected wethers. In Experiment 1, the deer developed clinical signs at 138 days after first contact and then died. OvHV-2 genes by polymerase chain reaction (PCR) and fluorescent antibodies to Alcelaphine herpesvirus-1 were detected in the affected deer. Moreover, sequences of PCR products (423bp), obtained by amplification of materials from the sheep and from the affected deer, coincided. These results clearly confirmed that the sheep was a carrier of OvHV-2, and that this virus had induced SA-MCF in a deer. In other experiments, no OvHV-2 infection occurred in deer and cattle during the 6-18 months periods of contact, though viral genes were detected in the nasal swabs and white blood cells of the sheep. To our knowledge, this is the first report on successful experimental transmission of MCF from OvHV-2-infected sheep to deer.