aliquots were prepared and stored at — 20 C. An initial titration of the virus employing one sheep per tenfold 

 dilution was performed employing 1.0 ml., s.c. Virus was detected in the 10" but not in the 10~ 3 dilution. 



A second titration of the virus blood was performed after 79 days of storage and virus was again detected in 

 the 10" 2 dilution. 



The third and last trial was performed after the virus had been stored for 364 days at —20° C. Bluetongue virus 

 was not detected in the 10 or 10 dilutions of the inoculum. The undiluted blood was not inoculated. A 

 hundredfold reduction ot virus titer had occurred during the storage in heparinized blood frozen at —20 C. 



Bluetongue virus in heparinized sheep blood and mouse brain suspensions was stored at —78° C. without gross 

 loss of potency. However, no precise titrations of BT virus stored in this manner were performed. 



Discussion 



The work of previous investigators was confirmed in that BT virus was propagated in lamb kidney cell cultures. 

 However, the detection of the virus by immunofluorescence was not a consistent finding. In addition, other primary 

 cell cultures and cell lines more readiiy available the year round did not grow the virus. It was concluded that the 

 isolation of BT virus by cell culture from viremic sheep blood was not a reliable diagnostic technique. However, 

 preliminary inoculation of the CE by the intravenous route or baby mice intracerebrally may be used to adapt the 

 virus. A subpassage to a suitable cell culture may then be performed. 



The irnmunofluorescent technique may be used to great advantage on the infected cell cultures since it has a 

 broad spectrum reactivity and the conjugate will stain all strains of the virus. 



Although the BT virus in sheep blood was not inactivated by freezing at —78 C. for 48 hours, the virus was 

 adversely affected by storage at —20 C. for 1 year. Thomas (14) has recommended rapid freezing in an alcohol dry 

 ice bath, storage at -78 C. and rapid thawing for the preservation of BT virus. 



In view of the stability of the virus at ambient temperatures, the use of OPG solution for the collection of 

 blood specimens for virus isolation is a useful procedure, particularly since intravenous inoculation of the CE 

 remains the method of choice for initial propagation of the virus. 



REFERENCES 



1. Boulanger, P., Ruckerbauer, G. M., Bannister, G. L., Gray, D. P. and Girard, A. Studies on Bluetongue. III. 

 Comparison of Two Complement-Fixation Methods. Canad. J. of Comp. Med. & Vet. Sci. 31, (July, 1967): 

 166-170. 



2. Bowne, J. G. and Jochim, M. M. Cytopathogenic Changes and Development of Inclusion Bodies in Cultured 

 Cells Infected with Bluetongue Virus. Am. J. Vet. Res. 28 (July, 1967): 1091-1105. 



3. Carbrey, E. A., McDaniel, H. A., Sherman, K. C, and Lee, L. R. An Investigation of the Etiology of Bovine 

 Glossitis Respiratory Complex Employing Specimen Material and Known Agents. Develop. Studies Conducted 

 by Diagnostic Services, NADL, FY 1968. ARS 91-81 (Dec, 1969): 26-28. 



4. , Stewart, W. C, Kresse, J. I., and Lee, L. R. Technical Aspects of Tissue Culture, 



Fluorescent Antibody Technique. USLSA, Proc. 69th Ann. Mtg., Lansing, Michigan (1966): 487-500. 



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 U.SA.H.A. (1971): 629-648. 



6. Fernandez, M. V. Isolation and Propagation of Bluetongue Virus in Tissue Culture. Am. J. Vet. Res. 20 

 (1959): 398-408. 



7. Haig, D. A., McKercher, D. G., and Alexander, R. A. The Cytopathogenic Action of Bluetongue Virus on 

 Tissue Culture and Its Application to the Detection of Antibodies in the Serum of Sheep. Onderstcpoort. J. 

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8. Mengeling, W. L., Gutekunst, D. E., Fernelius, A. L., and Pirtle, E. C. Demonstration of an Antigenic 

 Relationship Between Hog Cholera and Bovine Virus Diarrhea Viruses by Immunofluorescence. Canad. J. of 

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