Virology

Submission Guidelines for Virology

Sample selection, as well as proper collecting and handling of samples, is of critical importance prior to the submission of samples for virology. Please consider the following points.

1. Pertinent information concerning history, premise ID, herd size, owner's name, numbers and ages of animals involved, vaccinations, etc., should accompany the samples on the submission form. When possible, be specific as to the types of viral examinations being requested to avoid unnecessary charges.
2. Appropriate samples should be selected after the probable diagnoses have been considered. See individual testing pages for a listing of suggested specimens for each virus. If your virus of choice is not listed, please call the ADDL for instructions.
3. Swabs, fecals, or tissue samples taken as aseptically as possible from animals in the early stages of clinical disease are best. Most viral infections are cleared approximately 5 days after onset of signs.
4. Samples should arrive in the laboratory as quickly as possible in a chilled state (32-34°F). Insulated containers with ice packs are suitable for this purpose. Avoid fluctuating temperatures and slow freezing. The level of virus in tissue (when it arrives at the laboratory) is always less than at the time of collection.

Tissue Submissions

1. Postmortem samples should be taken immediately after the animal has died.
2. Organ tissues, to be evaluated separately, should be taken aseptically and packaged individually in plastic bags.
3. Segments of gastrointestinal tract (2-4" segments) should be submitted intact (unopened) and tied off with string or twine. Intestines or contents should not be placed in the same plastic bag as other organ tissues.
4. Additional tissue is required when more than one examination (e.g., VI, FA, EM) is requested.
5. Selection of the area of larger organs, such as lung, spleen, and brain, is often important. In tissue with marked changes, higher levels of virus are generally found at the active edge of a lesion. Necrotic centers of lesions or areas of normal tissue are often non-productive specimens for virus isolation, but may still yield a positive FA result.

Swab submissions

1. Submit only Dacron (polyester) swabs in transport media. Cotton swabs or swabs designed for bacterial culture may still be accepted, but will likely have a much lower chance of containing recoverable virus.
2. We recommend the BD Universal Viral Transport swabs. Sterile saline is acceptable if viral transport vials are not available. Please note that the liquid is saline on the submission form.

Electron Microscopy (EM)

1. Fresh, chilled fecal or cecal content samples are ideal for EM. Scabs, swabs and tissue may also be acceptable if enough viral particles are present.
2. Do not freeze samples prior to submission, doing so may break apart the virus particles making them hard to identify.

BVD PI Testing

Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae, the genus pestiviruses. There are several members of the genus and two types of BVD viruses. All of them are highly infectious and of economic importance to the livestock industry. If a fetus becomes infected in utero with BVD virus in the early stages of pregnancy, the newborn calf will become persistently infected (PI) without immune response to this virus for the rest of its life and will shed BVD virus in the herd, infecting other animals. If a PI animal is not identified in the herd, it will be a source of infection. Elimination of BVD from the herd requires removing PI animals.

There are several assays available to identify PI animals: Polymerase chain reaction (PCR), virus isolation (VI), and antigen-capturing ELISA (ACE). The ACE is a rapid diagnostic tool that can identify PI animals in the herd. Serum, whole blood, and ear notch samples are suitable for PI animal testing by ACE for BVD viral antigen.

1. Serum and whole blood sample considerations
   1. Only serum from precolostral newborn calves or calves older than 3 months are suitable for ACE testing.
   2. Maternal BVD antibodies, passed to the calf in the first 24 hours of colostrum intake, can interfere with the ELISA resulting in false negative results in PI calves. Ear notches may be tested from any age of calf.
2. Ear notch tissue (EN)
   1. Please notify the Virology Laboratory 24-48 hours prior to submitting large numbers (>50) simultaneously.
   2. Ear notches should be taken with a sharp ear notching tool suitable for adult swine. Baby pig ear notches, punches, and other cutting and punching tools are NOT recommended; the sample they provide is too small for an accurate test.
   3. It is strongly recommended that ear notchers be disinfected in 10% bleach after each sample is collected. Remember to then rinse off the disinfectant in plenty of clean water. Residual disinfectant will yield a false negative result. Do not vaccinate or tattoo animals at the same time ear notches are taken as this will also interfere with testing.
   4. Ear notches must be fresh and the samples must be approximately 1cm x 1cm (3/4 x 3/4 inches) in size. For short-term storage (1-2 days), samples can be refrigerated at 39°F; for long-term storage, samples can be stored at -4°F or colder.
   5. Avoid testing scabby or frostbitten ears.
   6. Do not put samples in formalin.
   7. Samples should be submitted fresh and chilled.
   8. Package individually in snap cap tubes (12x75 mm). These can be obtained from the following vendors: Fisher Scientific at https://www.fishersci.com (catalog number 14-959-2A) or VWR at https://us.vwr.com/store/ (catalog number 60818-419).
      • Blood collection tubes or other tubes of this diameter are acceptable. If sending for PCR, please send in RNase and DNase free tubes.
      • Do not use Whirl Pak bags to submit samples.
      • Failure to submit in appropriate tubes, may result in delayed turnaround time.
   9. Number tubes 1,2,3, etc., with animal identification to match information on accession form. Numbers should be clearly marked and legible.
      • Please utilized CF.755 Multiple Animal Continuation Form.xlsx from the ADDL Forms page to type animal and ID information when submitting large groups.
        • Please include a printed copy with the sample submission.
        • Please also send the file electronically to addl@purde.edu.
   10. A PCR test for pools of up to 25 ear notches (5 serum samples) is av.ailable.
       • Submit individual fresh samples in 5 ml sterile RNase and DNase free tubes, labeled with the animal identification, and a completed ADDL submission form. Specify that you are requesting the pooled BVD PCR.
       • Do not pool samples prior to submitting.
       • If the pooled sample is positive, ear notches or serum samples in that pool will be automatically re-tested individually by antigen capture ELISA.