Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.
We investigated the feasibility of using FTA filter cards for the storage of bursas of Fabricius containing infectious bursal disease virus (IBDV) and for IBDV detection by reverse transcriptase (RT)-polymerase chain reaction (PCR), and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. The FTA card is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBDV was inactivated upon contact with the FTA as shown by the inability of the virus to be propagated in embryonating chicken eggs. Viral RNA in minced bursas or stamped bursas could be amplified by RT-PCR (VP2 gene fragment, 248 base pairs) after storage on FTA for at least 15 days at room temperature or 8 mo at -20 C. Analytical sensitivity of the test was between 0.5-5 ng of RNA template or 5 x 10(1) mean tissue culture infective dose (TCID50)/FTA spot. Detection rate of IBDV in domestic clinical samples collected on FTA or collected by the non-FTA standard procedure was 36.7% and 41.7%, respectively, which represents 88% agreement. Detection of IBDV from FTA cards inoculated with bursal tissues in the laboratory or in the field was 36.7% and 37.1%, respectively. Detection of IBDV from FTA samples when the cards were inoculated with bursal tissues and sent through customs into the United States was 32.9%. Analysis of the amplified products showed that molecular characterization of IBDV by RFLP or nucleotide sequencing is feasible in bursas stored on FTA at 25 C for 1-3 mo or at -20 C for at least 8 mo. The use of FTA for the collection of bursal tissues and simultaneous inactivation of IBDV allows the movement of specimens within the United States and also from outside the United States in compliance with federal regulations and in a manner adequate for molecular characterization.
Moscoso
Hugo
H
Poultry Diagnostic and Research Center, Department of Population Health, The University of Georgia, Athens, GA 30602-4875, USA.
Alvarado
Ivan
I
Hofacre
Charles L
CL
D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D002060:Bursa of Fabricius / Q000821:virology*; D002645:Chickens / Q000821:virology*; D005374:Filtration / Q000295:instrumentation*; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000302:isolation & purification*; D010209:Paper; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000295:instrumentation / Q000662:veterinary*; D038301:Virus Inactivation
D012367:RNA, Viral