Genotyping and RAMALT testing for chronic wasting disease in cervids: Information for cervid producers
Chronic wasting disease (CWD) infection is confirmed through detection of the CWD prion agent in brain and/or lymphoid tissue. Owners may choose to have their cervids genotyped and/or have the recto-anal mucosa-associated lymphoid tissue (RAMALT) tested by third-party laboratories to identify potentially infected cervids for their own herd management.
Like all screening tests for CWD, there are considerable limitations to the information that a RAMALT test result can provide. Below is a list of frequently asked questions about RAMALT and genotyping that may help producers determine if they would like to incorporate these tests into their herd management practices.
What is RAMALT testing?
RAMALT testing involves taking a biopsy of a small band of lymphoid tissue just inside the anus of the live cervid. This tissue, called RAMALT, contains lymphoid follicles in which the CWD prion may be found if the cervid is infected with CWD. The tissue is then fixed and stained, and a veterinary pathologist views the lymphoid follicles to see if they have picked up staining attached to a CWD antibody. This test is called immunohistochemistry, or IHC. A minimum of six lymphoid follicles must be present on the slide for the test result to be meaningful.
What is genotyping?
Genotyping is also known as DNA testing. It is the process of determining the genetic make-up of an individual animal by examining the animal's DNA sequence. In the case of genotyping for CWD susceptibility, analysis of the animal's DNA will determine the genetic variation in the gene that encodes normal cellular prion protein. The development of CWD in a given animal may be affected by how effectively its cellular prion protein gene resists conversion to an abnormal CWD prion.
How does genotype affect susceptibility to CWD?
Research has shown that genetic variation does not provide resistance to CWD, but may affect how quickly the disease progresses once the animal is infected. Animals with more susceptible genotypes have a shorter incubation period for CWD and develop clinical disease more quickly. This information may be used by cervid owners when stocking or culling their herds.
How do the species and genotype of the cervid affect RAMALT test results?
The time between infection and appearance of CWD prion in the lymphoid tissue varies depending on the cervid's species and genotype. For example, some elk in the pre-clinical stage of infection will test positive in the brain but do not have detectable levels of CWD prion in the retropharyngeal lymph nodes. Such an elk may yield a false-negative RAMALT test. Elk with the gene 132LL are considered to have the longest incubation period for CWD. As a result, an animal may be infected for a significant period of time before the prion can be found in its lymphoid tissue. This may also result in a false-negative RAMALT test.
White-tailed deer, on the other hand, accumulate CWD prion in the cranial lymphoid tissue (such as the retropharyngeal lymph node) before it can be detected in the brain. White-tailed deer with the gene 96GG show the earliest development of disease after infection, and therefore may have prions present in their lymphoid tissue shortly after infection. RAMALT testing on an infected deer of this genotype could yield positive results.
Can RAMALT testing be done without genotyping?
RAMALT testing as a herd management tool is most useful if the genotypes of the animals in the herd are known as well. For example, the interval recommended before repeating a RAMALT test to follow up on the health status of a herd after a CWD-positive animal has been culled will depend on the genotypes of the animals. If the interval is too short in a herd with a large proportion of resistant genotypes, RAMALT testing may yield false-negatives when there is in fact CWD infection present in the herd.
How is RAMALT testing performed?
The RAMALT biopsy technique is difficult. Because RAMALT is a thin band of tissue just inside the anus, an inexperienced sampler may harvest tissue of the anus instead. Also, the piece of tissue must contain enough lymphoid follicles (at least 6) to provide a representative sample of the RAMALT of the animal. If too few follicles are harvested and tested, a false negative may result. The finite amount of RAMALT tissue also necessitates careful sampling and handling as repeat sampling may not be possible (as compared to blood or fecal tests). The person obtaining the samples for RAMALT testing needs to be properly trained in the technique.
How is genotyping performed?
Genotyping can be performed using a blood sample. However, the use of genotyping and RAMALT as herd management tools requires a degree of expertise. A plan for interpretation and application should be put in place before sampling a herd.
Why does CFIA not use RAMALT as a screening test for CWD in an individual animal?
The long incubation period and the variability in progression of CWD infection among different species and genotypes described above means that many cervids in earlier stages of the disease will test negative. In some infected cervids, CWD will not be distributed to, or detected in, lymphoid tissue (such as RAMALT). For this reason, RAMALT sampling and testing is best used as a screening test to determine the CWD status of a herd rather than that of an individual animal. A negative RAMALT test does not confirm that a sampled animal is negative, but a positive test result is significant.
How will the CFIA respond to animals with positive RAMALT test results?
An animal with a positive RAMALT test result is considered to be a CWD suspect and must be reported to the CFIA under the Health of Animals Act because CWD is a reportable disease. A cervid is only classified as infected with CWD when Canada's National Reference Laboratory for CWD, in Ottawa, reports positive confirmatory results using at least two CFIA-approved CWD tests.
The third-party laboratory that has conducted the RAMALT test will be required to send tissues to Canada's CWD National Reference Laboratory in Ottawa for review. If the CWD National Reference Laboratory determines that RAMALT test results are positive, the CFIA will order the animal destroyed with compensation so that it may undergo post-mortem confirmatory testing for CWD. This action applies to any farmed cervid with a positive RAMALT test result, regardless of the herd's participation in a Voluntary Herd Certification Program (VHCP) for CWD. For more information on the CFIA's VHCP for CWD, see our herd certification page. A farmed herd that is confirmed to be infected with CWD is subject to a CFIA investigation and if the herd is not ordered destroyed, it may also be subject to provincial disease control requirements.
The CFIA will not order an animal with a positive RAMALT test destroyed with compensation if that animal is part of a herd that has previously been confirmed infected with CWD.
How can I arrange to have my herd RAMALT tested and/or genotyped?
Three third-party laboratories are currently approved by the CFIA to conduct RAMALT and/or genotyping of cervids.
|Animal Health Laboratory
Laboratory Services Division
University of Guelph
Building 89, 419 Gordon Street,
P.O. Box 3612
Guelph, Ontario N1H 6R8
Telephone: 519-824-4120 (ext. 54530)
|Genotyping of elk
Genotyping of white-tailed deer
|Prairie Diagnostic Services
Room 2604 Diagnostic Immunology Laboratory
52 Campus Drive
Saskatoon, SK S7N 5B4
|IHC on RAMALT samples from cervids|
Animal DNA Department, Forensic & DNA Services
335 Laird Road, Unit 2
Guelph, ON N1G 4P7
Telephone: 519-836-2400, ext. 7067747 or ext. 7067714
Fax: 519-836-2377 or 519-836-4218
|Genotyping of elk|
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