Bluetongue virus infection in pregnant ewes.

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.

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring.

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.

Temporal relationships of viremia, interferon activity, and antibody responses of sheep infected with several bluetongue virus strains.

Sheep had viremias that were first detected on day 3 (+/- 1) after infection with several strains of bluetongue virus (BTV) representing United States serotypes 10, 11, 13, and 17. Diphasic peaks of infectivity were attained on days 6 and 10 (+/- 2). Interferon (IFN) was first detected in serum samples on day 5 (+/- 1), and reached greatest concentrations on day 6 (+/- 2), which coincided with the first viremic peak; IFN concentrations then decreased toward zero by day 10 (+/- 2). Interferon peak concentrations induced approximately a 90% decrease in virus titer. The decrease in IFN concentrations by day 9 (+/- 2) corresponded with the second viremic peak on day 10 (+/- 2). Onset of the decrease in detectable concentrations of virus after the second peak of viremia corresponded to the initial detection of serum antibody to BTV by day 10 (+/- 2). Virus titer decreased and antibody production increased until approximately days 21 to 28, when the titers plateaued and virus was not detected. Febrile responses peaked on day 7 (+/- 1) during the peak viremic period. The WBC count was depressed at the time the virus titer increased, but returned to normal values while the sheep were still viremic. Diphasic viremias in BTV-infected sheep were attributed to induction of high concentrations of IFN concurrent with the first virus titer peak, followed by production of antibody to specific BTV strains and a subsequent reduction in viremia at the second virus titer peak.

Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs.

Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

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.

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.

Salivary gland homogenates from the vector Culicoides variipennis may aid in detection of bluetongue virus in chronically infected cattle.

Studies were conducted on 2 cows chronically infected with bluetongue virus (BTV) acquired in utero from their dam. In previous research, BTV had been isolated 4 times from 1 cow and 8 times from the other. BTV was undetectable between spontaneous febrile and leukopenic episodes and antibodies to BTV were not detectable in the serum. Previous work had shown that bites by uninfected females of the vector Culicoides variipennis (Coquillett) caused virus to recrudesce in the blood to detectable levels in a bull with a similar BTV infection. After 1 of the 2 cows became hypersensitive to the bites of the vector, the cows were inoculated with homogenized salivary glands from the vector in an effort to increase the viral concentration to detectable levels. Bites by uninfected vectors were used 9.5-48 hr after inoculation of salivary glands in attempts to recover BTV biologically. In 2 of 6 experiments, after inoculations of the glands, BTV was isolated and confirmed from some of the blood samples and from some pools of C. variipennis females that had blood fed on both animals.

Effect of bluetongue virus on reproduction in sheep and cattle.

Bluetongue virus (BTV) has been shown to be arbortigenic and teratogenic. After transmission of BTV under natural and experimental conditions the consequences of vertical transmission in pregnant sheep or cattle are variable. Factors that influence reproductive consequences are the stage of gestation, characteristics of the virus, source and concentration of virus inoculum, placentation, season and the method and route of infection. Reproductive consequences vary greatly in degree but in general include infertility, abortion, mummification of the fetus, stillbirths and congenital anomalies and dysfunctions in the live offspring. Immunological unresponsiveness, sporadic viremia and development of "late disease" were consequences of vertical transmission in offspring of infected cattle. Perpetuation of BTV through 3 generations in cattle was documented. The presence of BTV in semen of infected bulls has been demonstrated.

Bluetongue virus in pregnant elk and their calves.

Two pregnant North American elk (Cervus canadensis), in the 3rd and 4th months of gestation, were inoculated with bluetongue (BT) virus (BTV) serotype 11. The virus was not isolated from the blood of the cows beyond postinoculation day (PID) 8, but was isolated from bone marrow and spleen samples obtained at necropsy on PID 190. Although neither cow had overt clinical signs of BT infection, fluctuations in specific neutralizing BTV antibody titers indicated viral replication. However, in 2 attempts, BTV was not recovered biologically via bites of colonized Culicoides variipennis (biting gnats) with subsequent transmission of the BTV to sheep. Bluetongue virus was isolated from the elk calves at birth and before they nursed. These calves remained latently infected, and BTV was transmitted from each calf to sheep by bites of the biting gnats. Most of the BTV biological recovery attempts resulted in suspicious BT clinical responses in sheep, but without viral isolation. However, after challenge exposure with the homologous virus, 5 of 7 recipient sheep bitten by the gnats reacted with an intensified BT clinical response that indicated viral sensitization. One calf was born weak, never attained a healthy appearance, was latently infected with BTV, and had fluctuating BTV neutralizing antibody titers. The other calf was in apparently good health, was latently infected with BTV, and was immunologically tolerant to BTV.

Abnormalities and virus-like particles in spermatozoa from bulls latently infected with bluetongue virus.

Abnormalities were commonly observed in spermatozoa, and bluetongue virus (BTV) was isolated from semen of 2 known BTV carrier bulls and 2 of 4 BTV seropositive field bulls. The spermatozoal abnormalities ranged from a small cavity between the acrosome and nucleus with some involvement of the nucleus, to an enlargement of the cavity accompanied by vesiculation that could affect the entire acrosome. Virus-like particles were occasionally observed in the affected spermatozoa, but were present in all samples. A positive relationship was found between infectivity of semen samples from BTV latently infected bulls and the observation of abnormalities and virus-like particles in the heads of affected spermatozoa.

Bluetongue virus in bovine semen: viral isolation.

Vero cell cultures and embryonating chicken eggs were used for direct isolation of bluetongue virus from cattle blood and from semen samples. Cell culture and embryonating chicken eggs each were more effective than was the blood autograft inoculation of susceptible sheep with selected blood and semen samples. Evaluation of the cell culture technique indicated that the quality of the distilled water was the primary factor responsible for the increased sensitivity of the Vero cell cultures for the present blue-tongue viral isolations. Test results showed that urine was a poor specimen for viral isolation when assayed in chicken eggs. A comparison of tests for precipitating and complement-fixing antibodies to bluetongue virus indicated that the precipitin test was the more accurate of the two tests.

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 cattle: effects of vector-transmitted bluetongue virus on calves previously infected in utero.

Three of 7 principal calves, after a challenge of immunity exposure by bites of bluetongue (BT) virus-infected Culicoides variipennis, became latently infected with BT virus. These calves were born to heifers infected with the homologous virus by bites of C variipennis at 60 or 120 days' gestation. Latent BT virus infection was detected by isolation of BT virus from washed erythrocyte samples obtained from the calves at 57, 100 to 102, 200 to 202, 300 to 302, and 400 to 402 days after challenge of immunity and from 1 of the calves over 5 years after challenge of immunity. The 3 latently infected calves were healthy; 2 were immunologically competent and 1 was immunologically incompetent to develop detectable BT virus antibodies in their blood. Bluetongue virus infection was detected (by viral isolation) in 2 other principal calves during the challenge of immunity, but they were not considered latently infected. The latter 2 calves were immunologically incompetent to develop detectable BT virus antibodies.

Bluetongue in cattle: repeated exposure of two immunologically tolerant calves to bluetongue virus by vector bites.

A heifer and steer that were immunologically tolerant to bluetongue (BT) virus became immunologically competent after repeated exposures by bites of BT virus-infected Culicoides variipennis. Immunologic tolerance ended in the heifer at 25 months of age, after the 2nd exposure to the virus, and in the steer at 22 months, after the 4th exposure. High hemic concentrations of BT virus were detected in both animals after they became immunologically competent, but neither developed an overt BT clinical response. The steer died suddenly and extensive pathologic changes were observed at necropsy.

Overwintering mechanism for bluetongue virus: biological recovery of latent virus from a bovine by bites of Culicoides variipennis.

Bluetongue virus (BTV) was biologically transmitted by the bites of colonized Culicoides variipennis gnats to recipient sheep from a Hereford bull with latent infection. Four biological recoveries of BTV were mediated over 2 years by multiple feedings of the vector during a 4- to 72-hour interval. Initial stimulation by gnat bites at 0 hour permitted biological recovery of BTV by gnats that fed at later intervals. The 4th biological recovery of the virus from the bull clearly indicated a vector-mediated viral recovery mechanism in which initial vector bites at 0 hours stimulated a "showering" of BTV into the blood stream of the bull. The BTV carrier bull developed no overt signs of illness during these studies. The pertinent BTV assay and serologic results for the 4 1/3 years of the bull's life are summarized in this report.