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Rectal Biopsies May Hold Clues to Eradication

By Alan Harman

A new live-animal test to detect scrapie is being applied to goats and research is continuing into goat genes that could offer resistance to the degenerative disease at Colorado State University, Washington State University, and Pullman. This test and research are results from eight years of work coordinated by the Animal and Plant Health Inspection Service (APHIS) on an accelerated national scrapie eradication program to eliminate the disease from U.S. goats and sheep.

The new test, known as the rectal mucosal-associated lymphoid tissue (RMALT) test, is based on the now used third-eyelid test. Developed by the Agricultural Research Service and Washington State University (WSU), the third-eyelid test has been used by APHIS and state veterinarians since 2002 as an official test to detect scrapie in sheep.

Microbiologist Katherine O’Rourke and molecular biologist Tom Truscott obtain tissue from a goat for use in a new test for scrapie. (Photo by Peggy Greb.)
Microbiologist Katherine O’Rourke and molecular biologist Tom Truscott obtain tissue from a goat for use in a new test for scrapie. (Photo by Peggy Greb.)

Researches said it has been hard work because transmission routes, progression, and genetic underpinnings of scrapie in goats are poorly understood. Low occurrence rates, underreporting, and the inconvenience and cost of tissue testing make eradication challenging. In goats and sheep, scrapie causes tremors, lip smacking, weight loss, a hopping gait, and other peculiar symptoms. Scrapie-afflicted animals cannot be cured and eventually die.

The test involves snipping a tiny piece of lymphoid tissue from the animal’s nictitating membrane, or third eyelid, staining it with antibodies, and examining it under a microscope. Lymphoid tissue is used because it tends to collect malformed proteins called prions, which are thought to cause scrapie.

Researchers in Norway and Scotland modified the method to use lymphoid tissue in the lining of the animal’s rectum. The rectal biopsy is quick to perform and relatively painless to the animal—thanks to a dab of local anesthetic.

"Rectal biopsy also allows for more repeat samples from an individual animal when needed," said ARS microbiologist Katherine O’Rourke, a member of the scrapie research team that includes APHIS Veterinary Services (APHIS-VS) and Wildlife Services, the National Park Service, Colorado State University, and the Canadian Food Inspection Agency.

APHIS-VS approved use of the RMALT test as a scrapie-detection tool after large-scale validation trials comparing its 87% accuracy to third-eyelid biopsies and postmortem examinations of tissue from infected sheep. Additional work on the accuracy of the test in goats is under way.

APHIS has incorporated the RMALT test into the national scrapie eradication program. Current users are mainly federal and state veterinary personnel.

A key facet of scrapie prevention in sheep flocks involves use of selective breeding to increase the number of sheep with the version of the prion protein gene—dubbed R171—that confers resistance.

For now, goat producers don’t have that option. Despite searches by ARS and WSU scientists at Pullman, as well as at other labs, the R171 prion gene version has never been found in goats. But there are several different gene variants in goats, some of which might confer resistance to scrapie. No one has confirmed genetic resistance in goats thus far, but some tantalizing leads are emerging.

"In sheep, the discovery of resistance genes was key to developing a broadly accepted eradication program," O’Rourke said. "If scrapie is found in a flock, only the genetically susceptible sheep are removed, allowing the producer to maintain quality animals. As we learn more about goat genetic resistance, we hope the same approach can work for them."

Towards that end, ARS Pullman geneticist Stephen White is leading a team of ARS and university scientists to characterize the prion protein gene of goats and identify important gene variants in individuals and breeds. The Pullman team has so far examined the prion protein gene sequences from 446 goats representing 10 breeds, eight of which have never been genetically characterized for their potential scrapie response.

The team’s analysis found four gene variants—R143, S146, H154, and K222—in the genes of Boer, Nubian, Saanen, Toggenburg, and a few other goat breeds. These gene variants were relatively rare or absent in animals that developed scrapie in previous outbreaks, which suggests these gene variants might help the animals in some way.

Pullman geneticist Stephen White studies goats with a version of the prion gene that might confer increased resistance to scrapie. (Photo by Peggy Greb.)
Pullman geneticist Stephen White studies goats with a version of the prion gene that might confer increased resistance to scrapie. (Photo by Peggy Greb.)

A fifth variant (M142) was found mainly in Alpine and Toggenburg goats, and it is known to delay incubation of scrapie from infection to clinical disease. More work is needed to demonstrate true resistance for any of these genetic variations, notes the team in a paper published in the journal Genetic Selection Evolution.

In related work, the Pullman scientists are monitoring six goats from a Michigan farm with a known history of scrapie infection. In March, two of the infected goats, named Nutmeg and Meeko, gave birth to three kids, providing the scientists with an unprecedented look into parent-to-offspring transmission of scrapie and inheritance of genes affecting the animal’s response to the disease.

"We learned that—unlike in sheep —the goat placenta is not a very reliable indicator of the scrapie status of the dam," O’Rourke said. "We’ll do some more research to determine why this is, and how it affects transmission in a herd."

Genetic testing revealed Meeko’s kid is genetically susceptible, which allows for monitoring of scrapie’s onset and development during the animal’s life. Nutmeg gave birth to twins—one genetically susceptible and the other with a variation that may eventually prove to confer resistance.

The ARS Pullman team is collaborating closely with APHIS-VS to formulate a strategy aimed at helping the U.S. goat industry to eliminate scrapie. The current effort takes a multi-pronged approach that includes early detection through slaughter surveillance and reporting of clinical suspects, flock management and selective breeding in sheep, scrapie-free flock certification, and producer outreach and education.

"In support of the eradication effort led by APHIS and industry, ARS will continue to do research on genetic resistance, diagnostic testing, and transmission modes," O’Rourke said. "Prevention is always a more desirable route than removal of infected animals or exposed animals. So research on resistance genetics and transmission modes will be especially important contributions."

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