Submission Guidelines

• Please completely fill out the Submission Form.
  • Please fill in as completely as possible; if an answer is unknown, please indicate so.
• Fresh tissues and carcasses should be shipped with sufficient numbers of ice packs, positioned so as not to crush smaller carcasses or samples. Avoid packaging formalin-fixed tissues with ice packs, as this can affect microscopic evaluation of these tissues.
• Whenever possible, avoid shipping severely autolyzed carcasses. See the Carcass Quality Guide for more guidance.
• In general, we strongly prefer whole carcasses to allow us to make a more complete and holistic evaluation; however, we recognize that some circumstances do not permit this and there may be rationale for necropsying on-site (such as more rapid collection into formalin for highly valuable samples).
• If a necropsy was performed prior to submission, formalin-fixing tissues of interest as soon as possible helps decrease post-mortem changes that affect microscopic evaluation.
  • Tissues should be collected in formalin immediately, unless tissues are cold/frozen, in which they should first be allowed to thaw prior to placing in formalin.
  • Ideally, the ratio of formalin to tissue should be approximately 10:1. For shipping, tissues can be fixed first, then later transferred to a leak-proof container with a smaller volume of formalin.
  • Submitting both formalin-fixed tissues and fresh/refrigerated/chilled/frozen tissues is ideal.

• Ideal samples include formalin-fixed and fresh, refrigerated/frozen:
  • Brain (fresh and formalin-fixed) or head (fresh)
  • Lung
  • Spleen
  • Heart
  • Kidney
  • Liver
  • Gastrointestinal tract (e.g., stomach compartments, small and large intestines)
  • Any lesions of interest including those listed above as well as other tissues, e.g., adrenal gland, reproductive organs, skin, bone marrow, tongue, skeletal muscle, lymph nodes near sites of any lesions, limbs, eyes, etc.
• Please submit any photos of lesions or significant findings from the field or necropsy, when able.
• For cases where there is concern for toxins, extra fresh liver, kidney, stomach contents, and brain are recommended samples. In some cases, samples collected from the environment may be relevant (e.g., any suspicious bait piles or tainted carcasses). If a specific toxin is of concern, you may wish to speak to a diagnostician before shipment, as they may be able to recommend the ideal samples to collect for that case.
• If submitting cassettes with formalin-fixed tissues:
  • Label with appropriate formalin-proof pen and ensure writing is legible.
  • Cassettes should be submitted in secure formalin containers and formalin should completely cover the cassettes.
  • Screw-top lid formalin jars are preferred and parafilm or similar material can help seal the tops and prevent leaking.
  • Jars should be surrounded with absorbent material (e.g., paper towel) in case of leaks and should be kept in a sealed plastic bag.
  • Include a reference list of cassettes submitted and what tissues they contain.
• If there are any questions about what samples to submit, please call or email SCWDS for assistance.

• Legal cases require special considerations and communication prior to submission.
• A chain of custody form should be initiated by the person collecting samples and/or carcasses in the field (e.g., law enforcement officer, agency biologist, etc.) and contact your law enforcement division for assistance during the field investigation.
• SCWDS is not specifically trained or recognized as a wildlife forensic laboratory, but we will document the case and all findings to the best of our ability.
• If using SCWDS, contact SCWDS personnel to consult prior to collection and submission.
• If using a veterinary forensic laboratory, such as the National Fish and Wildlife Forensics Laboratory (USFWS; https://www.fws.gov/law-enforcement/clark-r-bavin-national-fish-and-wildlife-forensics-laboratory) or a state veterinary diagnostic laboratory, we recommend you consult with their staff prior to collection and submission.
• We will retain samples associated with cases that are indicated as legal upon submission for up to three years from the time of submission; please update us as to the status of the case and when any associated samples can be discarded.

Ideal samples are the entire head or both retropharyngeal lymph nodes and obex (fresh prefered over formalin-fixed). For deer, retropharyngeal lymph nodes alone can be tested but to gain more information about stage of infection, obex would be needed. For elk, obex and retropharyngeal lymph nodes (or the entire head), should be submitted.

SCWDS does not routinely test captive cervids for CWD. Exceptions can be made for unusual circumstances after direct communication with a diagnostician prior to sending

• Please specify whether the case is a clinical/diagnostics case or for a special/research project.
• Please specify the anatomic location where the parasite was found (e.g. within the small intestine, within the back muscles etc.) and any other pertinent information.
• For submission of just a parasite for identification, the parasite should be submitted in 70% ethanol.

What viruses do we test for?

Routine hemorrhagic disease (HD) testing is for epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). SCWDS can also test for cervid adenovirus if warranted based on on field signs or postmortem findings.

What species do we test?

SCWDS tests all North American cervids (e.g. white-tailed and mule deer, elk, moose), pronghorn, bighorn sheep, and other species as necessary. For other species not listed, please contact the diagnostic service or SCWDS HD laboratory for further guidance before shipment.

What tests are run and when?

In peak HD season (July through October), samples are tested via RT-PCR and virus isolation is performed on positive samples. Serotyping is also performed on virus isolation positive samples. During non-peak seasons, testing will be limited to RT-PCR on cases with appropriate field signs or necropsy findings that are suspect HD casesy.

What samples are preferred for testing?

Preferred samples are pieces of fresh (unfrozen) lung and/or spleen for EHDV, BTV, and cervid adenovirus. Lymph node or whole blood in anticoagulant can also be submitted if the other two samples are unavailable. Bone marrow is not preferred but can be used in specific circumstances. Samples of approximately golf ball to tennis ball size are preferred.

How should samples be stored?

Samples should be stored refrigerated and shipped with ice packs. If long delays (e.g. weeks) between collecting and shipping are anticipated, samples can be frozen; however, this may impact our ability to isolate viruses in cell culture.

How much does testing cost?

Typically, all hemorrhagic disease testing is performed at no charge for wildlife agencies, regardless of SCWDS membership status in support of the long-standing participation of all states in the SCWDS Annual HD Questionnaire. Charges for non-member states does occur occasionally.

General information:

• Please follow the most recent instructions sent in SCWDS emails, as testing capabilities and protocols may change over time.
• Full carcasses or oral/cloacal swabs in appropriate media (see below) can be submitted for highly pathogenic avian influenza virus (HPAIV) testing at SCWDS.
  • Samples from cases that test positive via laboratory testing at SCWDS will be sent to the USDA National Veterinary Services Laboratory (NVSL) for confirmatory testing. Due to the very close matching of SCWDS and NVSL test results thus far, submitters will be notified of NVSL test results only if they disagree with previously reported SCWDS results.
• Please specify if the location of the sick/dead individual(s) is from an area/county with known HPAI cases, and if so, what species have had confirmed detections.
• For mass die-offs suspected to be due to HPAI based on field findings and known history in the area, we recommend shipping 1-3 of the freshest carcasses per outbreak site. Please review most recent SCWDS emails regarding HPAIV for further instructions.
• Mammals will be tested on a case-by-case basis primarily based on history and pathology findings. If there is particular concern or relevant clinical history, please include this information on submission paperwork.

Swab recommendations:

• Polyester, non-wood, standard swabs

Viral transport media recommendations:

• PrimeStore™ MTM Molecular Transport MediaSDP (Manufacturer:  PrimeStore™ MTM LH105)
• SCWDS protocol:
  • 4 ml cryovials containing 2 ml of chilled viral transport media (VTM) consisting of brain heart infusion media (Becton Dickinson and Co., Sparks, MD, USA) supplemented with antibiotics [penicillin G (1000 units ml−1), streptomycin (1 mg ml−1), kanamycin (0.5 mg ml−1), gentamicin (0.25 mg ml−1) and amphotericin B (0.025 mg ml−1)] (Sigma Chemical Company, St Louis, MO, USA)

Media storage (before sample collection):

• The media tubes can be held at -20C (regular freezer temperature) indefinitely.
• The tubes can be thawed and are stable in the fridge at 4C for about one week.
  • The vials can be returned to the freezer until needed if not used within a week.

Preferred swab samples:

• Swab the oropharyngeal cavity and cloacal cavities separately (each with own swab) and place both swabs into the same media vial.
• Cloacal and/or oropharyngeal swabs submitted in separate vials is also acceptable.

Sample (HPAI swab) storage:

• Tubes should be kept cold (NOT frozen) and shipped to SCWDS within a few days.
• If shipping is delayed, tubes should be frozen at -80C (laboratory freezer).

• Ideal samples for testing include fresh or frozen liver, spleen, and bone marrow; only a small sample (1/2 cm3) of each is needed for molecular testing.
• We routinely screen all submitted wild turkeys whenever full carcasses or the appropriate samples are submitted.

• For smaller animals (e.g., foxes), the entire carcass can be submitted.
• For larger animals (e.g., bears) where the entire carcass cannot be submitted, please submit large samples of fresh skin for testing. Pieces from multiple sites and areas of transition from abnormal to more normal skin are ideal.

• The ideal sample is the entire rabbit carcass, especially in states where tularemia is of concern as there is zoonotic risk to opening the carcass outside of a laboratory setting. Otherwise, a fresh liver sample is preferred (entire liver or a portion is fine).

• If there is human exposure (e.g., bite/scratch), consultation with local public health officials will determine where the carcass should be sent. Please do this prior to contacting SCWDS. In many cases, the head and/or carcass should be sent to a public health laboratory for rabies testing. Once there is a negative rabies result, the carcass (if saved) can then be shipped to SCWDS.
• If you choose to submit to SCWDS for complete carcass evaluation, please make sure the history clearly describes any and all potential human and/or domestic animal exposure, and the decision of the consulted public health department. If unknown, please follow up with whoever initially reported the case.
• For cases of neurologic mammals without human exposures, submission of the entire carcass (e.g., bats, foxes, raccoons, skunks) or of the head (e.g. deer) is recommended.

• We recommend submitting the entire carcass so we can closely examine the skin and viscera for suspicious lesions to sample, which can be subtle. In cases where the whole carcass cannot be submitted (e.g., sample from a live snake), submission of fresh skin lesions (e.g., affected scale(s)) is recommended. If the sample is large enough, half can be submitted fresh and half can be submitted in 10% neutral-buffered formalin. If no formalin is available, or the sample is small, fresh tissues are fine.

• Submissions are tested on a per case basis. Please provide as much information as possible. Of particular importance to note are possible toxins in the area (e.g., rodenticide usage, pest control methods, herbicides, etc.), the environment in which the animal(s) was found (e.g. farm, urban area, etc.), size of area affected, number of species (and quantity of each) affected, timeline of observations, and any suspected toxins of specific concern. Toxicology testing is very expensive and no test can detect all toxins; narrowing the list of toxins of concern helps the SCWDS diagnostics team and collaborating toxicologists decide what tests to prioritize. This is especially helpful in cases with smaller animals or one or few animals affected, as there may not be sufficient fresh tissue to run multiple tests.
• If multiple small animals (e.g., passerines) are affected, submit multiple birds so that enough liver and other organs are available to make up a large enough pooled sample for toxicology. Contact the diagnostician to help determine an appropriate number of carcasses, in cases with large scale die-offs.
• For brevetoxin testing, whole carcasses or samples of liver/kidney/lung/stomach contents/plasma/serum are ideal.
• Eagles are routinely screened for heavy metals. Anticoagulant rodenticide and other toxicology tests are done on a case-by-case basis. If there is consistent history or clinical concern, please make sure to include this information on the submission form.
• If suspicious samples in the environment are found (bait piles or other potential consumed items), please bag separately and send these as well.

• Ideally, submission of the entire bat carcass is recommended. If unavailable, additional samples to submit include swabs of the skin over the muzzle and wing membranes (for Pd detection only; not disease). Swabs can be stored in dry, individual containers/vials and kept refrigerated or frozen prior to sending.
• SCWDS does not test environmental or guano samples for Pd.