Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe.

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.

Rapid identification of bluetongue virus by nucleic acid hybridization in solution.

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with 35S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 microliter reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.

Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus.

Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1-25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6-48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 micrograms of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates.

In vitro testing of a potential retroviral vector for producing transgenic livestock.

The amphotropic murine leukemia virus (MLV) has been shown to infect mammalian species other than mice. If this virus infects and expresses genes in cells of livestock species (cattle, sheep, and pigs) it has potential for use as a vector to produce transgenic livestock. Because the gene-injection technique for producing transgenic animals is inherently inefficient, our laboratory was interested in identifying or constructing retroviral vectors capable of infecting livestock embryos. The infectivity of an amphotropic MLV-based vector for ovine, bovine, and porcine cells was tested. Experiments were also conducted to test the ability of the amphotropic MLV promoter, compared with known strong promoters, to express genes in cells from these species. Results indicated that amphotropic MLV infects and expresses genes efficiently in porcine cells and is, therefore, a potential vector for producing transgenic pigs. Infection was not detected in cells from adult bovine and ovine species; however, low levels of infection, with subsequent gene expression, were detected in cells derived from bovine embryos.

Sequence relationships of United States prototype and wild-type bluetongue virus RNA genomes investigated by northern blot hybridization analysis.

The 10 double-stranded RNA (dsRNA) genome segments of various isolates of bluetongue virus (BTV) were separated on a polyacrylamide gel, denatured in NaOH, and blotted onto 2-aminophenylthioether paper. Blotted dsRNA segments were detected, using radioactive probes, a cloned copy of DNA 70% fragment of genome segment 7 of BTV-17, whole genome BTV-17 copy DNA, or whole genome BTV-17 dsRNA. These probes detected sequence diversities in different isolates of BTV and these diversities are discussed in relation to the serotype and the electrophoretic migration patterns of the isolates.

Multiple bluetongue virus-cloned genetic probes: application to diagnostics and bluetongue virus genetic relationships.

A shotgun-cloning method incorporating all 10 bluetongue virus genome segments can simultaneously produce complete and partial copies of any of the genome segments. We report here 4 different cloned probes derived from 3 genome segments and individually defined by different hybridization recognition capabilities. One probe hybridized strongly with all 5 United States prototype strains of the 5 different bluetongue virus (BTV) serotypes existing in the United States and, as such, is a strong candidate for a broad BTV diagnostic probe in the United States. Another probe derived from genome segment 2 of BTV-17 hybridized only with the BTV-17 prototypic serotype, thereby demonstrating serospecific hybridization diagnostic potential. The implications for diagnostic and genetic relationship studies on BTV, using various genetic probes, are discussed.

Bilateral ureterocystostomy in a 450-kg horse with ectopic ureters.

A 450-kg yearling Clydesdale filly was determined to have bilateral ectopic ureters. The resulting incontinence caused severe malodorous perineal dermatitis. Bladder capacity was measured at 800 ml. The urethral sphincter lacked tone, and the horse was seen to urinate in a normal manner only 2 or 3 times a week. A midline celiotomy was performed, and the ureters were identified by cannulation from the ectopic openings. The ureters were ligated, and the cut ends were anastomosed to the dorsal bladder surface by an extravesicular end-to-side technique. A partial thickness seromuscular layer of the bladder was sutured over the ureters in a cranial direction from the anastomosis site for 15 mm. This fixed the ureters to the dorsal surface of the bladder and protected the anastomosis site from tension. After surgery, the horse urinated n a normal manner many times a day. Urinary incontinence continued, but gradually improved. Bladder capacity increased over 13 months to 4.3 L. Surgical (urethral extension) and medical (phenylpropanolamine and estrogen) treatments were instigated to increase urethral sphincter tone. Urinary incontinence continued to improve and, at 11 months after surgery, incontinence was negligible, and the perineal dermatitis had healed.

Bilateral wedge ostectomy on an 18-month-old llama with severe bilateral carpal valgus.

An 18-month-old llama was admitted with severe (45 degrees and 40 degrees) bilateral carpal valgus. The llama had grown normally until it was 6 months old, when the carpal deviations had commenced. Radiography revealed abnormalities at the distal ulnar physes. Premature closure of these physes or abnormal distal ulnar growth may have been the cause of the carpal valgus. The owners requested surgical correction of the condition. Medial wedge osteotomies of each radius were performed 3 weeks apart. Internal fixation of the osteotomized radial bones was accomplished with small right-angled T plates, and the limbs were placed in full-limb casts for 3 weeks and tube casts for 3 more weeks. Both limbs healed in straight alignment, and the llama was able to walk and run normally. Dorsal subluxation of the right radiocarpal joint was noticed after the operation. This was believed to be caused by the uncorrected dorsal bowing of the distal portion of the radius, which had occurred secondary to the severe carpal valgus.

External fixation repair of a displaced comminuted radial fracture in a juvenile camel.

A young female camel had a complete comminuted midshaft fracture of the right radius. The fracture was repaired by external coaptation, which involved 2 full-limb fiberglass casts for 15 weeks. A Thomas splint was placed around the second cast for 12 weeks. The fracture healed in nonaligned configuration, and although lameness was substantial after the fracture had healed, the camel's breeding potential had been salvaged. This successful outcome is an indication that a high-limb fracture in a camel bone (in this case a radius) may be managed by external coaptation and repair by callus formation.

Postpartum partial cystectomy through the vagina in a mare with everted partially necrotic bladder.

The bladder of a 750-kg Clydesdale mare had everted through the urethra into the vagina immediately after parturition. The bladder was reinverted into the peritoneal cavity by an attending veterinarian, but 4 days later, the bladder was everted again in the vagina. The mare was able to void urine through both ureters, which could be seen in the mucosal surface of the bladder. The everted bladder had become edematous and could not be reinverted through the urethra. A considerable portion of the fundus was necrotic. The mare was administered xylazine epidurally to induce perineal analgesia, and the necrotic portion of the bladder was resected and healthy bladder tissue was opposed with a double layer of simple continuous sutures. The urethral sphincter was longitudinally incised through the vaginal mucosa to allow reinversion of the bladder through the urethra. A purse-string suture inserted in the urethral opening decreased the urethral diameter and prevented recurrence of the condition. An inflated Foley catheter was maintained in the bladder for 5 days. The mare recovered normal urination after the catheter was removed.

Arthroscopic removal of a palmar radial osteochondroma causing carpal canal syndrome in a horse.

A 5-year-old Arabian stallion with moderate effusion in the right carpal canal and intermittent lameness in this limb was diagnosed to have an osteochondroma projecting from the distal portion of the radius into the carpal canal. oral phenylbutazone treatment over the next 3 years allowed the stallion to continue its show career. Right forelimb lameness returned at that time, and ultrasonography revealed the osteochondroma impinging on the dorsal surface of the deep digital flexor tendon. The owner elected to have the osteochondroma surgically removed. The horse was anesthetized, and the carpal sheath was distended with balanced polyionic solution. A 4-mm arthroscope was inserted into the carpal sheath, and the osteochondroma projecting into the sheath was identified. The osteochondroma was removed by use of a Ferris-Smith bone rongeur, which was inserted into the carpal sheath through a stab incision over the osteochondroma. The effusion in the carpal sheath and the lameness resolved by 2 months, and the horse was returned to training 4 months after surgery.

Full-length diaphyseal sequestrum as a consequence of segmental transverse fractures of the large metatarsal bone in a calf.

A 1-week-old calf was treated for fractured right metatarsus; however, at 3.5 months of age, the fracture site again became unstable. Radiography disclosed segmental transverse metatarsal fractures with an isolated, full-circumference diaphyseal sequestrum. Arteriography and nuclear scintigraphy confirmed the isolation and necrosis of the diaphyseal segment. Diaphyseal sequestra usually develop as cortical or partial circumferential necrotic fragments. The finding of a full-length diaphyseal sequestrum was unusual.

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.

The clinico-pathological effect of bluetongue virus serotype 20 in sheep.

Fifty-four Merino crossbred sheep were inoculated with bluetongue virus serotype 20 (BTV-20) by the intravenous, subcutaneous and intradermal routes. BTV-20 was successfully transmitted by Culicoides (Avaritia) spp. No. 5 to two additional sheep. Clinical and pathological effects were studied. In the artificially infected sheep, clinical signs were observed after an incubation period of 6 to 10 days and consisted of pyrexia, oral and subcutaneous hyperaemia mild oedema of the ears, face and lips, and coronitis. The major internal pathological changes were petechial and ecchymotic haemorrhages in the tunica media of the pulmonary artery near its junction with the heart and mild haemorrhage and mild oedema in the intestines, coronet, lips, cheeks and ears. Viraemia was detected between day 2 and day 14 post inoculation. The two sheep infected by insect transmission were mildly affected and became viraemic between 16 and 19 days after transmission. No deaths occurred and under experimental conditions BTV-20 caused only mild disease in housed sheep. To date there has been no reported outbreak of natural bluetongue infection in Australia. Compared to other serotypes BTV-20 appears to be of low pathogenicity in sheep.

Experimental infection of bulls and cows with bluetongue virus serotype 20.

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.

Bluetongue virus serotype 20: experimental infection of pregnant heifers.

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.

Epizootiological study of bluetongue virus infection in California livestock: an overview.

An epidemiologic program was undertaken in California to study bluetongue virus (BTV) infection in domestic livestock. The study was designed to determine: a) prevalence of BTV infection, b) serotypes of BTV actively causing infection, c) seasonality of infection and d) species infected. A total of 8,751 cattle, 14,639 sheep and 4,785 goats were tested over the 3 1/2 year study. Serologically, 41% of the cattle, 42% of the sheep and 21% of the goats were positive. Virologically, 2.4% of the cattle, 1.4% of the sheep and 0.7% of the goats were viremic. One BTV isolation was made in April (sheep) and 3 in June (1 each from sheep, cattle and goats); the remainder of the BTV isolations (a total of 359) were made in the months of July through early December. No isolations were made from January through March. Four serotypes of BTV (10, 11, 13 and 17) were isolated from all species tested (sheep, goats and cattle) and Culicoides variipennis. The serotypes isolated from C. variipennis correlated with the serotypes isolated from livestock in given areas. Multiple serotypes were isolated from single herds, flocks and individual animals on single given days. In addition to multiple serotypes being isolated, extensive heterogeneity in the electrophoretic mobility of the RNA genome segments was observed. These different migration patterns (electropherotypes) were observed between and within serotypes. No single serotype could be identified by a given pattern. No clinical disease was associated with BTV infection of cattle. Clinical disease was observed in infected sheep; however, BTV was also isolated from flocks with no overt clinical signs of disease. No reproductive problems could be associated with BTV in cattle in this endemic study area; however, BTV infection of pregnant sheep appeared to be associated with abortion and birth of dummy lambs in certain flocks.

Strain-dependent virulence characteristics of bluetongue virus serotype 11.

Two strains of bluetongue virus (BTV) serotype 11, UC-2 and UC-8, were identified by the electrophoretic migration pattern of their genomic RNA segments on polyacrylamide gel electrophoresis. Significant differences in virulence of these two viruses could be demonstrated by subcutaneous inoculation of newborn mice. No signs of disease were observed in mice infected with UC-2. Mice infected with UC-8 died of a severe necrotizing encephalitis, which resembled lesions in bovine and ovine foetuses infected with BTV.