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.

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.

A hallmark of clinical bluetongue virus (BTV) infections in sheep is multisystemic inflammation, which is often most readily noticeable in the oral mucocutaneous tissue of whitefaced breeds of sheep.6J0 Arborvirus infections of the endothelial cells in inflamed cutaneous sites and in visceral organs is do~umented.~ It is thought that infection of endothelial cells, which results in lysis or in direct damage to the microvasculature, is a major pathophysiologic mechanism that accounts for increased vascular permeability and the classic clinical signs of bl~etongue.~ Although perivascular infiltrates of mononuclear cells are described in BTV infection~,~ the phenotype of the infiltrating lymphoid cells has neither been examined, nor has their role in the development of lesions and protection been addressed. Recently, we reported changes in peripheral blood T lymphocytic subsets during acute bluetongue infections in sheep and cattle.4 Of particular interest were the shifts in the OvCD4 and OvCD8 T lymphocytic subsets, which occurred in the peripheral blood of sheep. These occurred at two time periods-approximately l week after infection, at the nadir of the characteristic transient leukopenia, and approximately 2 weeks after infection, early in the convalescent period, when BTV-specific cytotoxic lymphocytes were identified in the thoracic duct lyrn~h.~ Such changes may be a reflection of alterations in lymphoid traffic in sites of viral replication. The purpose of this investigation was to examine the changes in the dynamics of lymphoid populations in inflamed oral mucocutaneous tissue, at time points that corresponded to the shifts observed in the peripheral blood, subsequent to BTV infection in sheep. Each of four BTV-seronegative yearling Columbia x Rambouillet sheep (Nos. 14) were inoculated with approximately 2 to 4 x 10s median chicken embryo intravascular lethal doses (CEIVLD,,) of BTV serotype- 10 (BTV- 10) inoculum.4 Equal amounts ofthe inoculum were injected subcutaneously in each ear and intradermally into bilaterally shaved areas of cervical skin. Two contact sheep (Nos. 5,6) each received an inoculum of an equal volume of uninfected, allogeneic, whole sheep blood. Rectal temperatures, complete blood counts, and occurrence of mouth lesions6 were recorded daily. Two sheep were euthanatized at 7

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.

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.

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.

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.

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 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.

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.

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 (bt) viremia in Saanen goats was assayed by inoculating blood into embryonating chicken eggs. High-titer viremia consistently occurred in Saanen goats inoculated with either BT 8 or 63-66B strain of bt virus. Low-titer viremia consistently occurred in Saanen goats inoculated with 4 other strains of bt virus. Virus was detected as early as postinoculation day (pid) 1, increased to a peak on pid 6 or 7, and then gradually decreased, but was still detectable as late as pid 21. Goats did not develop overt signs of illness, and 13 of 18 goats inoculated had group-specific bt viral precipitin antibodies in their blood serums on pid 21. All goats had antibodies 21 days after the challenge viral inoculation was given.

Intravascular inoculation of embryonating chicken eggs with sonically treated blood samples from bluetongue (bt)-suspect sheep and cattle was found to be equivalent to sheep inoculation for the isolation of virus. There were 32 and 31 viral isolations made in eggs and sheep, respectively, out of the 40 isolations made by either or both systems. Nine isolates (7 of cattle origin and 2 of sheep origin) that were obtained by egg inoculation were test negative in sheep, and 8 isolates (6 of cattle origin and 2 of sheep origin) that were obtained by sheep inoculation were test negative in eggs. The infective agents isolated in eggs were subpassaged on primary lamb kidney cell cultures and identified by bt virus-specific fluorescent antibody (fa). Some of the isolates were also identified by fa tests on heart and kidney tissues of infected embryos and by inoculation in susceptible sheep.

The attenuated live-virus (TC-83) Venezuelan equine encephalomyelitis (vee) vaccine was serially back passaged, using 5 groups of 5 horses each. During back passage, clinical responses of horses increased in frequency and severity, with a corresponding higher viremia. Two horses died, one at 34 days after inoculation (4th back passage) and one at 36 days (1st back passage), but a specific cause of death was not determined. Young adult guinea pigs did not die after inoculation with back-passaged virus or with the same virus after suckling mouse brain passage to enhance the titer.

Bluetongue (bt) virus was isolated from naturally infected cattle with signs similar to those of foot-and-mouth disease. Three cattle, with no serologic or virologic evidence of foot-and-mouth disease, bovine virus diarrhea, infectious bovine rhinotracheitis, or vesicular stomatitis, were purchased and studied clinically, virologically, and serologically. Two were pregnant heifers: one aborted, and bt virus was recovered from the fetus; the other had a live calf of dwarflike conformation and with other congenital deformities. The 3rd, a 3-year-old cow, was extremely emaciated and apparently harbored the bt virus. Evidence indicated that: (1) bt viral strains isolated in these studies have low pathogenicity and immunogenicity for sheep, and (2) bt virus can be isolated from cattle throughout the year as opposed to the usual summer and fall seasons for sheep.