IBV Sequencing Service - Procedures
for IBV Inactivation and Shipping, USDA Import Permit, and Fees
Instructions for Phenol inactivation of Avian Infectious Bronchitis Virus (IBV) and Packaging for Shipment
1. Inoculate the suspected IBV isolate into 9-to-11-day-old embryonating chicken eggs via the allantoic cavity. Harvest the allantoic fluid 48-72 hr after inoculation. Place isolate in a freezer (-20C or colder) if the allantoic fluids are not to be phenol-treated for shipment for longer than a day or two. If frozen, thaw the fluids and proceed with the phenol inactivation (see 2. below).
NOTE. IBV isolates that produce typical chick embryo lesions (stunting, curling and kidney urates) give the best results. Please check to make sure the suspected IBV isolate that you intend to send for sequencing produces typical embryo lesions. Also, if possible, test the isolates for the presence of avian adenovirus type 1 by agar gel precipitin (gel diffusion) prior to submission. Avian adenoviruses are common in the respiratory tract of commercial chickens and produce embryo lesions that are highly similar to IBV.
2. Place 2-3 ml of allantoic fluid in a labeled 15 ml polypropylene plastic snap-cap tube, 17 x 100 mm (Falcon 2059) (or equivalent) and add an equal volume of saturated buffered phenol (pH 4.3-4.5). The tube and cap must be polypropylene! (other plastics may dissolve in phenol!). Make sure the cap is tightly secured. Wrap the top of the tube and cap with parafilm to prevent leakage. Note, the phenol must be molecular biology grade suitable for nucleic acids. The acidified (pH 4.3) phenol is highly recommended! We recommend Fisher (catalog no. BP1751I-100) but any high grade phenol will work. DO NOT USE DISINFECTANT GRADE PHENOL! IT WILL DESTROY THE ACTIVITY OF THE IBV NUCLEIC ACID.
3. An approved aluminum-lined biohazard mailing container should be used for shipping the IBV sample. An approved aluminum-lined biohazard mailing container may be obtained from Fischer Scientific, (Fischerbrand biohazard mailers, catalog no. 03-523-2 will accommodate the Falcon tube (17 x 100 mm) listed above). If aluminum containers are not available, use a disposable plastic 50 ml conical centrifuge tube with screw cap (VWR Scientific Products cat. no. 21008-242).
4. Place the parafilm-sealed IBV allantoic fluid sample in the approved aluminum-lined biohazard mailing container and place in a styrofoam box. Seal the styrofoam box with reinforced mailing tape. No ice packs or dry ice are needed.
5. Place a copy of the import permit and an original letter from you, the sender, confirming that the allantoic fluid was phenol-inactivated and tape both on the outside of the package in a sealed envelope so that they may be examined by US federal inspectors. A copy of the permit is required! Contact Dr. Gelb by e-mail or FAX (see below) for a copy of the permit. Include your e-mail and FAX number so we may send a copy of the permit to you.
Ship by Overnight/Next day Delivery (Federal Express is the Preferred
6. NOTIFY DR. GELB BY E-MAIL (JGELB@UDEL.EDU) OR FAX (302-831-2822) OF YOUR INTENTION TO SHIP A SAMPLE(S) AT LEAST TWO WEEKS BEFORE SENDING IT.
Cost of S1 Genotyping of Avian Infectious Bronchitis Virus (IBV) (In US Dollars)
1. PCR Product Cycle Sequencing (To identify IBV genotype, perform predicted
amino acid similarity analysis vs. other IBV genotypes in GenBank
2. Shipping, if necessary. (Overnight delivery from USA port of entry
to University of Delaware)........$50.00
List of Countries from Which Inactivated Avian Infectious Bronchitis Virus (IBV) Samples May be Obtained