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PUT YOUR TA ARGET IN THE CROSSHAIRS. WE’LL DO THE REST.
Pneu n D a r t .c o m 8 6 6. 2 9 9.DA R T
Whethe r it ’s a 250 - cla s s a nimal or a n e ntire he rd, you ne e d Pne u - Da r t, a nd our half- ce ntur y of expe r ie nc e. O ur A me r ic a n - made d pate nte d te chnolo g y is de signe d to re duce trauma a nd promote ide al delive r y. T hat ’s why ce r vid ra nche r s, wa rde ns, a nd othe r s k now us a nd use us. Pne u - Da r t. O utsta nding in our f ield.
CONTENTS
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FAWN FEATURES 22
Bloody, Bloated Fawns by Dr Joe Ables, DVM, MA
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Nutrition of Fawns: Birth to Weaning By Michael L. Schlegal, PhD, PAS, Dipl. ACCAS- Nutrition - Purina Wildlife
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Prepare For Fawning Seasons by NADR
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Killer Fawns Disease: One Farmer’s Report on an Important Discovery by Cleve Tedford
FEATURES 29
Deer & Coronavirus by Shawn Schafer
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Fusobacterium Necrophorum by Cervid Solutions
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Have You Converted to SNPS by NADR
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SNPs vs STRs: Who Cares and Why The Change? by NADR
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Have You Heard of Humic Acid? by Bobby Deeds, Wildlife Specialist - Record Rack
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What Is The Cervidae Health Research Initiative?
DEPARTMENTS 6
president’s Message by Sam Holley
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Calendar of Events
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Executive Directors Message by Shawn Schafer
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nADeFA Board of Directors
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CLF Board of Directors
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new Members
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Legislative report by Capitol Hill Consulting
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Treasurer report by Hank Dimuzio
PRODUCTS & EQUIPMENT 134 Advertiser’s index
By Samantha M. Wisely, Professor of Wildlife Ecology and Director of the Cervidae Health Research Initiative 83
Fungus Among Us: The Potential for
COVER:
Biting Midge Control by Robert JR Ewing and Lee W. Cohnstaedt 89
The Evolution of Wildlife Anesthesia by BAM Pharma
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Medgene Labs’ EHDV Vaccine Update by Ashley Petersen
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Accurate Genomic Predictions for Chronic Wasting Disease in U.S. White-Taild Deer
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Fawn Photographed in the forest of Toronto
Failure of Fallow Deer to Develop CWD When Exposed to a Contaminated Environment and Infected Mule Deer
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Ensuring the Safety of Natural Deer Urine Products By Davin Henderson, CWD Evolution
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Humic Acid Degrades CWD Prions in Soil! By Dan Harrington
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PRESIDENT’S MESSAGE
Proud To Be Part Of This Exceptional Association During These Trying Times NADeFA Members Unite Wow, what a ride! The conference is coming together quickly, speakers are set, meals planned, booths laid out, new deer farmers seminar, antler competition... then the rug is no longer under us and we are scrambling in the other direction trying to inform everyone that we had to cancel because of a virus? We quickly changed directions and organized an online auction to help us with our annual fundraiser. Postcards are mailed out and everything is set until we had to cancel last minute because of a virus? Now we are faced with contacting the members with another cancelled event that was out of our control. In the process of contacting our members, it became clear that we are made up of an exceptional group of individuals. The response from members with their donations was unbelievable. Often times in the face of the member being laid off from work and the future uncertain. The decision was made by the board that each family or person that had registered should be contacted in person by either a board member or Shawn. As these calls were made the true value and strength of our organization became apparent, it is our members and the relationships they have formed over the many years. The formation of these relationships cannot be affected by a virus or social distancing. While the virus is still around as of the printing of this magazine, it is also evident that NADeFA is getting stronger because of it! Our election this year brought us three new board members, Alan Hochstetler (Ohio), Chris Ezell (Oklahoma), and Dan Jennings (New York). Doug Roberts, Jason
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Edmonson and I were also elected to continue serving on the board. We are excited to begin working with the new board members in the coming months. This year we also had two long standing board members that decided to retire. Skip West served three years as Board President and had served on the board and CLF board for nine years. Kevin Grace served on the board for over 18 years. Thank you and we appreciate all your years of service and dedication to NADeFA. The NADeFA website and Facebook pages continue to change and evolve. The CLF once again has its own page on the website. We are always looking for information to be shared on these sites so if you have any articles or events please forward them to either Shawn or myself. We will soon be adding links directly to our sponsors from the homepage. The sponsors are also gaining a dedicated page where they will be able to share a brief description of their ranch or products. Our sponsors have always been there supporting us when needed and we need to remember to support them when possible as well. It is our hope that all of our members and their families stay safe and healthy through all of these trying times. Once we have made it past these unchartered waters, we will resume making plans to gather and share in our great association. u See you all soon, Sam Holley NADeFA President
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NADeFA CALENDAR Submit your events to info@nadefa.org nADeFA MiSSion
Southeast Trophy Deer Association Sping Fling July 10-11 Orlando, FL
North Dakota Elk Growers Summer/Fall Meeting August 22 2020 Devil's Lake, ND
MnEBA Summer Picnic July 25 Pequot Lakes, MN
Deer Breeder Corp Annual Convention August 28-29, 2020 San Antonio, TX
Exotic Wildlife Association Congressional Fundraiser August 6-9, 2020 Boerne, TX Texas Deer Association Annual Convention August 14-15, 2020 San Antonio, TX
To foster a greater association among people who raise deer for commercial purposes, NADeFAÂŽÂ is dedicated to the promotion of deer farming and ranching as an agricultural pursuit and serves its members through its educational programs and publications and by providing leadership in setting and maintaining quality standards.
PDFA Whitetail Auction September 2020 Pennsylvania 2020 USAHA Conference October 17-21, 2020 Nashville, TN
For more information on NADeFA and / or to become a member please call (330) 454-3944 or visit www.nadefa.org Deer Farmer is published quarterly by the North American Deer Farmers Association. Graphics and pre-press production for North American Deer Farmer is provided by Verso Media group.
Columnists & Contributors Dr Joe Ables, DVM Hank Dimuzio Sam Holley Shawn Schafer Capitol Hill Consulting Cervid Solutions Article submission, photography, reader's letters, story ideas and other correspondence should be sent to: NADeFA 4501 Hills & Dales Rd NW Suite C Canton, OH 44708 tel (330) 454-3944 fax (330) 454.3940 All rights reserved. Photocopying, reproduction or quotation prohibited without permission from the publisher. Unsolicited material cannot be acknowledged or returned.
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EXECUTIVE DIRECTOR’S MESSAGE
We Are Humbled By The Support We Have Received From Members March 13, 2020 was a tough day for me, after months of preparation and planning for our 2020 NADeFA “Annual” conference, the board of directors met to discuss Governor Holcomb’s ban on all gatherings of more than 250 people in the state of Indiana. There really wasn’t much discussion as our options were rather limited. I had called French Lick Resorts earlier that day after the ban was enacted and they basically said unless we can cut our event in half that we had no options but to cancel. I will admit that initially I was having mixed feelings about if we had made the correct decision, but as the days progressed, so did the Coronavirus and so did the bans, stay at home orders, and resulting cancelations. As the saying goes the rest is history, or should I say the crazy history we are living right now, as this will impact our lives for years to come. We tried to look at alternative dates, but rescheduling quickly became a
nightmare as so many other events were being postponed, that finding availability without conflicting with other industry events just wasn’t possible. We tried to maintain the fundraiser by switching to a live online video auction and removing the large items that would have to be shipped, but as the stay-at-home orders increased, many of our members who have another job or business outside of their deer farm, found themselves or their spouses (or both) without jobs, so it was decided that we should postpone the online sale until the stay-at-home orders are lifted and everyone is allowed back to work. I have said on more than one occasion, that it was best to cancel our conference because if anyone in the French Lick area ever came down with Covid-19 our industry would get blamed, and I didn’t want the headlines to say “Deer Farmers Spread Disease”! Now while that may be funny as
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(Member of another member business/farm has to be same business name - No vote, no registry) Practitioner $35 (No vote or registry) Student $35 (No vote or registry)
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it seems like we are always being blamed for spreading CWD, it also makes you step back and compare how the two are actually very similar (especially in how they have been handled) – bans on deer farms, bans on high fence hunting, closed borders, predictions of major death loss, no one truly knows where they started or how long they have been around, if a death doesn’t actually happen in your family or to someone you know then we tend to downplay the disease as it really isn’t that bad! That last one is probably making you say “hey what the heck does Shawn mean by that”? Let me explain, you see both diseases and the programs put in place to control them are hard to evaluate because the more successful the program the less disastrous the disease looks. Now don’t get me wrong, while I believe either disease is capable of causing death, I don’t believe they will cause death in everyone (or every deer), I also believe there has been a lot of over-reaction to each disease and that with the proper biosecurity and hygiene practices each can be managed without destroying livelihoods’. With that, I would like to step back to our canceled conference and the educational sessions we had planned which would fall into the management I was referring to in the previous paragraph. Dr Justin Greenlee and Dr Eric Nicholson from USDA/ARS at the National Animal Disease Center in Ames, Iowa, were going to give reports on their ongoing CWD research projects including genetic resistance, disease shedding and progression. Dr Davin Henderson was going to give an update on his live CWD testing company, CWD Evolution, and using amplification tests such at RTQuIC for not only live testing, but also to test urine for the scent industry, and to evaluate prions in other items such as soil and semen. Dan Harrington and Dennis Simpson were going to present on their findings with Humic Acid as a management and prevention tool. A presentation I was looking forward to the most was Dr Chis Seabury, Texas A&M, as he discussed his recently published paper “Accurate Genomic Predictions for Chronic Wasting Disease”, for those of you that have not been following the work of Dr Seabury or that may not totally understand the whole CWD resistance efforts, the best way I can explain it is I believe this will be the game changer that our industry has been waiting for to put CWD behind us once and for all. I would not even Spring 2020
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attempt to get into the nut and bolts of his research, as it is far too complex for me, but I can say that Chris has developed a computer program to evaluate 123,987 SNP’s of the whitetail genome and how they are associated with the prion genes thereby providing the necessary foundation for developing a genomically-estimated CWD eradication program. Watch for more information to be coming early this summer. Moving away from the CWD front, Ashley Petersen, Medgene Labs, South Dakota, was going to report on their new EHD 2/6 multivalent vaccine that is now available. If you are interested in protecting your deer herd against EHD 2 and 6 you can call Medgene at 605-690-2316. Ashley could have explained it far better than I, but I can tell you this is not an autogenous vaccine which our industry has been limited to for so many years, and they have the lab results to prove it develops and antibody response. I would like to say how proud I am to be working for such a great industry and the people that it entails. As the NADeFA board of directors and myself reached out to those members and vendors that were registered for the conference to notify them of the cancelation and their pending refunds, we were repeatedly honored and humbled by the donation of their registration funds to support NADeFA and the wellbeing of the industry! In closing I would like to offer my condolences to the Robbie Troyer family. I was reflecting back trying to remember how many years I have known Robbie, and it brought back a memory from 2013 when I was at the Pennsylvania Fall Auction and fundraiser, and they were raffling a nice hunting rifle, which Robbie was lucky enough to win. I will never forget when he walked up to me and introduced himself and said that he would like to donate that rifle to support NADeFA and the deer industry! Truly a great man! Please keep the Troyer families in your thoughts and prayers. u Shawn Schafer Executive Director (651) 212-1315 North American Deer Farmer
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NADeFA
Executive Committee
President
2nd Vice President
Sam Holley (2023) Oak Ridge Whitetail Adventure 7143 Noble Rd Windsor, OH 44099 440.636.3040 sam@huntoakridge.com
Jason Edmondson (2023) 3 E Whitetails 155 Edmondson Ln Alabaster, AL 35007 205.296.0062
Dr. Hank Dimuzio (2022) LedgEnd Farm 1288 Munger St. Middlebury, VT 05753 Phone: 802.388.8979 Fax: 802.388.8979 Email: ledgendeer@comcast.net
3rd Vice President
Executive Director
1st Vice President
John Whetstone (2021) Whitetail Syndication 25595 CR 54 Nappanee, IN 46550 574.773.2179 whetstonebrothers@gmail.com
Jacques deMoss (2022) Winter Quarters Wildlife Ranch LLC 10113 W. Farm Rd. 124 Bois D'Arc, MO 65612 Phone: 337.322.2569 jacques.demoss@gmail.com
Board of Directors
Fred Huebner (2021) Circle H Whitetails 2575 Iowa Keokuk North English, IA 52316 319-530-7824 circleh@netins.net
Chris Ezell (2023) Tulsa View Whitetails 7134 W 420 Rd, Chelsea, OK 74016 918-633-4013 chrisezell@rocketmail.com
Daniel Jennigs (2023) Jennings Brothers Farms LLC 143 Beach Hill Road, New Ashford, MA 01237 413-822-1040 dan.jennings02@gmail.com
Alan Hochstetler (2023) Double H Whitetails 9850 Winesburg Rd, Dundee, OH 44621 elkaldiesel@yahoo.com 330-466-1514 Brad Heath (2021) Orion Whitetails W13055 Akron Ave Plainfield, WI 54966 715-335-6080 brad@orionwhitetails.com
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Steve Munz (2021) Galaxy Whitetail Solutions 847 S Main St Wildwood, FL 34785 352-266-4270 galaxysteve@msn.com Doug Roberts (2023) Conquest Deer Farm 8399 Bristol Rd Davison, MI 48423 810-241-9554 droberts@conquestdeerfarm.com
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Juan Lino Garza (2021) Ranchos Garza US/MEX 2121 Sunset Lane Mission, TX 78572 210.393.5233 jlgarza1@aol.com
Rich Meech (2021) Trophy Rack Productions 45 Larch Ave. Menahga, MN 56464 218-564-5090 star@wcta.net
NADeFA is dedicated to the promotion of deer farming and ranching as an agricultural pursuit and serves it’s members through educational programs and providing leadership in setting and maintaining quality standards.
Mark Cobb (2022) Gobblers Ridge Exotic Animals 215 Eastwood Rd. Ravenswood, WV 26164 304-532-4514 or 304-532-5304 mark.cobb@gmail.com
Brad Farmer (2022) Farmer's Fallow Deer 120 E. Robinson St. Viola, KS 67149 Phone: 316.772.7592 BradShirlFarmer@sktc.net
Shawn Schafer Schafer Whitetail Ranch 1223 18th Ave. N.W. Turtle Lake, ND 58575 Phone: 701.448.2002 Cell: 651.212.1315 Email: schafer@nadefa.org
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NADeFA
Treasurer
Jesse Seltmann (2022) X 4 Ranch 18430 166th Rd Denison, KS 66419 785-845-1143 jbseltmann@hotmail.com
PHONE: 330.454.3944 | FAX: 330.454.3950 4501 Hills & Dales Rd NW, Suite C Canton, OH 44708
info@nadefa.org
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WinTEr 2019
CLF
Board of Directors
CLF Executive Committee Chairman: Mark Cobb Gobblers Ridge Exotic Animals 215 Eastwood Rd. Ravenswood, WV 26164 Phone: 304-532-4514 or 304-532-3304 Email: mark.cobb@gmail.com
1st Vice Chair: Brad Farmer 120 East Robinson Viola, KS 67149 Phone: 620-584-6635 Email: bradshirlfarmer@sktc.net
Treasurer: Dr. Hank Dimuzio LedgEnd Farm 1288 Munger Street Middlebury, VT 05753 Phone/Fax: 802-388-8979 Cell: 802-343-8848 NADEFA Cervid Livestock Foundation exists to serve the deer industry through educational, charitable and scientific purposes relating to deer farming and ranching and the use of deer products. The Cervid Livestock Foundation seeks to influence industry trends and assure a healthy and expanding industry. The Cervid Livestock Foundation's mission is to facilitate public education about the agricultural and economic value of raising deer. MISSION:
Educate the public as to the value and benefits of deer and deer products Disseminate information relating to the care and breeding of cervid species Conduct programs to support the education of deer farmers/ranchers regarding the deer industry and venison consumption Support scientific research Promote the expansion of the North American Cervid Industry
Mr. Ray Burdette El Canelo Ranch P.O. Box 487 Raymondville, TX 78580 Phone: 956-689-5042 Fax: 956-689-1089 Email: ray@elcaneloranch.com Mr. Sam Holley Oak Ridge Whitetail Adventure 7143 Noble Rd Windsor, OH 44099 Phone: 440-636-3040 Fax: 440-272-5325 Email: sam@huntoakridge.com Mrs. Carolyn Laughlin Hilltop Whitetails 9025 Bachelor Rd. NW Magnolia, OH 44643 Phone: 330-866-5421 Fax: 330-866-5851 Email: carolyn@nadefa.org Dr. J. Bradley Thurston Luke’s Run 2640 Ponderosa Road Spencer, IN 47460 Phone: 765-795-6406 Cell: 317-372-8493 Email: thurstonbrad@yahoo.com Mr. Skip West Circle W Whitetails 54301 S. 351 Rd Maramec, OK 74045 Phone: 918-671-8669 Email: skip@circlewhitetails.com
Current initiatives - The CLF and NADeFA sponsor a wide variety of educational activities: adult programs, youth programs, scholarships, scientific research, public awareness and marketing of deer products.
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George Courtney • 682-229-7008 • george.courtney12@gmail.com
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FAWNS
BLOODY BLOATED
» FAWN HEALTH
By Dr Joe Ables, DVM, MA Nothing is more frustrating than finding a sick fawn. Many questions run through our minds what is going on? Within the first few months of life the number one problem we face with fawns is G.I. problems. A complete physical exam is critical to solve the problem. It is most important to diagnose the problem rather than just treating without knowing the problem. There are tests available to diagnose problems with fawns by examining the feces. First of all, I am not a proponent for doing bacterial cultures in feces. Feces are contaminated and do not yield a pure successful diagnosis and bacterial isolation. Fecal floatation and examination for parasites in feces is the most common and 22
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most efficient way to produce a successful diagnosis. Fecal samples can be sent in to TVMDL at parasitologist under the request of parasite screening. Dr Craig at Texas A&M University is one of the top parasitologists in the nation. I have often heard Breeders speak of fecal samples yielding negative results. If you have fecal samples run at a local Veterinarian and the result comes back with negative, you can send the samples into TVMDL for further evaluation. One of the most common problems we encounter with ON NEXT PAGE 24»
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» FAWN HEALTH
« CONTINUED FROM PAGE 22
Adults typically do not suffer from Giardia nor coccidia fawns with bloody diarrhea is coccidia. For years we have and have been commonly found in their GI tracts. Only the treated coccidiosis with many different medications and still adult deer with weakened immune systems appear to have have less than 100% response to treatment. I have discovered problems fighting these protozoan parasites. Knowing that with multiple fecal examinations run by Texas Veterinary adults can be "carriers" of these protozoans, how can we Medical Diagnostic Lab were positive with coccidia but also prevent the transmission to fawns? Pregnant doe suspected had Giardia which has been left untreated due to everyone of having these parasites can be evaluated and treated with reaching for Corid thus with a response to therapy of 50%. approved medications a month prior to fawning to prevent How do we treat Giardia? The texts books tell us that transmission. fenbendazoles daily for 5 days have a proven to be effective. It is also imperative to do Necropsies on any dead fawns. Contact your Veterinarian to find the appropriate treatment Samples can be sent in to specifically isolate pathogenic strategy for your fawns. bacteria. Once the bacteria have been isolated a sensitivity 24
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test can be accomplished to discover what antibiotics the bacteria are sensitive to. Remember it is very critical to provide pre-and probiotics concurrently to fawns that are under antimicrobial therapy. Another common problem with experience with fawns is bloating. What causes fawns to bloat? One of the number one causes of bloating in fawns is “ileus.” Ileus is a $10 word for immobility of the intestine. When the small intestines discontinue to contract, air builds up within the intestines causing expansion of the intestinal wall. This forced pressure on the wall occludes(constricts) the blood vessels and prevents oxygen to the nerves, "paralyzing" or numbing the nerves as you have experienced for yourself when sitting Indian Style too long. Fawns can be "tubed" to help relieve the pressure on the rumen but not the small intestines or colon. Puncturing the left side of the abdomen with a needle only expels air from the rumen as well and can introduce and bacteria to the abdominal cavity producing peritonitis, dead fawns. A few over the counter drugs methods commonly used in veterinarian medicine to relieve GI gas are mineral oil and simethicone (Malachon drops). Mineral oil helps break down the surface tension of gas bubbles and helps
decompress the intestines, similar to the use of Malachon drops (simethicone). Other brand names under this category are Gas X, Phazyme, Beano, etc. Also, ask your Veterinarian about “Prokinetics.” Prokinetics are simply what their name implies, forward movement. They help stimulate the nervous system contraction from the stomach to the direction of the colon, which helps push the gas out. Contact your Veterinarian for the correct dosages. Ok, ready for a recap? Bacteria fermentation can cause gas which causes bloat. The pressure on the intestinal wall can cause terrible pain and is often referred to as colic. Responding quickly to these cases yields the most promising results. Parasites (especially Coccidia) and certain types of bacteria can cause bloody diarrhea. Antibiotics do not treat Protozoan and anti-protozoans do not treat bacteria. A thorough diagnostic and treatment plan is critical for correct therapy. Get ahold of your Veterinarian now to have a plan in action prior to trouble. If you have any questions please feel free to call. u Dr Joe Ables, DVM, MA Enable USA Drjoeables@yahoo.com
®
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LEADING THE CERVID INDUSTRY FOR 37 YEARS
Why NADeFA? Eligible to participate in the North American Deer Registry Representation of all Deer Farmers at a Federal Level Receive 4 issues per year of the North American Deer Farmer Annual Membership Directory Industry Updates via email or mail Access to our Veterinary Health Line Take part in Annual Cervid Congress where industry issues and solutions are discussed Participate in Annual Conference including, trade show, education breakout sessions and auctions
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Do you own a Hunting Preserve or Ranch?: ____________________________________ Do you market Venison? ____________________________________________________ Scent Collection? __________________________________________________________ Species: __________________________________________________________________ Species: W = Whitetails, A = Axis, E = Exotics, F = Fallow, M = Muntjac, MD = Mule Deer, PD = Pere David, R = Reindeer, RD = Red Deer m YES
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» COVID-19 & CERVIDS
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DEER &
CORONAVIRUS By Shawn Schafer Executive Director of nADeFA
After the announcement of tigers in a zoo and some pet cats catching covid-19 from people, I have had several members ask “what about cervids?” I received a paper from a researcher that works closely with respiratory diseases such as TB, in it, they looked at the cell receptor that the current coronavirus binds to when it infects a host. They used known sequences that are in databases to predict which species have a high likelihood of being successfully infected with the coronavirus. Here is a quote from the paper: "We were surprised to find that all three species of Cervid deer and 12/14 cetacean species have high scores for binding of their ACE2s to SARS-CoV-2 S. There are 18 species of Cervid deer found in China. Therefore, Cervid deer cannot be ruled out as an intermediate host for SARS-CoV-2. While coronavirus sequences have been found in white tailed deer (53) and gamma coronaviruses have been found in beluga whales (54, 55) and bottlenose dolphins(56) and are associated with respiratory diseases, the cellular receptor used by these viruses is not known." The three deer species they looked at were white-tailed deer, reindeer and Pere David's deer. These three deer species were more likely to become infected than cats, which so far have been the domestic species most seen with any kind of clinical signs. ON NEXT PAGE» Spring 2020
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» COVID-19 & CERVIDS
« CONTINUED FROM PAGE 29
Most believe that the virus came from bats, went to an unknown intermediate host and then to humans. I am only sharing this, so if you or someone else that may be around your deer, develops Covid-19 you may want to take precautions around your animals to prevent any possible transmission just like you would other people. Here are the recommendations from the American Veterinary Medical Association Q: Should i avoid contact with animals, including pets, if i am sick with CoViD-19? You should restrict contact with pets and other animals while you are sick with COVID-19, just like you would
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restrict contact with other people. Although there have not been reports of pets becoming sick with COVID-19 in the United States, it is still recommended that people sick with COVID-19 limit contact with animals until more information is known about the virus. Have another member of your household care for your animals, if possible. If you have a service animal or you must care for your animals, then wear a cloth face covering; don’t share food, kiss, or hug them; and wash your hands before and after any contact with them. You should not share dishes, drinking glasses, cups, eating utensils, towels, or bedding with other people or pets in your home. Cats should also be kept indoors as much as possible. While we are recommending these as good practices, nADeFA.org
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there is no evidence to suggest that animals, including pets, that may be incidentally infected by humans are playing a role in the spread of COVID-19. Q: if i am ill with CoViD-19 are there special precautions i should take to prevent spreading disease, including when caring for my pet? If you are sick with COVID-19 you need to be careful to avoid transmitting it to other people. Applying some common-sense measures can help prevent that from happening. Stay at home except to get medical care and call ahead before visiting your doctor. Minimize your contact with other people, including separating yourself from other members of your household who are not ill; use a different bathroom, if available; and wear a cloth face Spring 2020
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mask when you are around other people or pets and before you enter a healthcare provider’s office. Wash your hands often, especially before touching your face, and use hand sanitizer. Use a tissue if you need to cough or sneeze and dispose of that tissue in the trash. When coughing or sneezing, do so into your elbow or sleeve. The AVMA recommends you take the same commonsense approach when interacting with your pets or other animals in your home, including service animals. You should tell your physician and public health official that you have a pet or other animal in your home. As a precaution, it is recommended that people sick with COVID-19 limit contact with animals until more CONTINUED ON NEXT PAGE»
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» COVID-19 & CERVIDS
« CONTINUED FROM PREVIOUS PAGE
information is known about the virus. So, if you are ill with COVID-19, have another member of your household take care of walking, feeding, and playing with your pet. If you have a service animal or you must care for your pet, then wear a cloth face covering; don’t share food, kiss, or hug them; and wash your hands before and after any contact with your pet or service animal. You should not share dishes, drinking glasses, cups, eating utensils, towels, or bedding with other people or pets in your home. Cats belonging to owners infected with COVID-19 should also be kept indoors as much as possible. While we are recommending these as good practices, there is no evidence to suggest that animals, including pets, that may be incidentally infected by humans are playing a role in the spread of COVID-19. u 32
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979-203-6735
info@D DanInjectDartGuns.com o
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» FAWN NUTRITION
nuTriTion oF
FAWnS BirTH To WEAning By Michael L. Schlegel phD, pAS, Dipl. ACAS-nutrition Sr. nutritionist, Wildlife and Small ruminant Technical innovation purina Animal nutrition LLC
Newborn fawns represent the start of a new generation, genetic improvement, and the future. Since there has been an investment in the doe and the buck, it is important to get fawns off to a positive start to see that return. The nutrition of fawns starts with the doe even before fawns are born during the last third of pregnancy and continues and through lactation. Two additional items to a successful fawn program include colostrum consumption and getting fawns started on feed. The greatest amount of fawn fetal growth rate occurs during the last third of pregnancy with approximately 67% of the birth weight gained during this period. Does that are malnourished during the last third of pregnancy will have fawns that are smaller and weigh less than if they received an adequate plane of nutrition. After fawns are born, one of the most critical management practices is to ensure that fawns consume colostrum within the first 24-36 hours after birth to provide passive immunity (the transfer of antibodies from the doe to the fawn). Fawns will typically nurse 36 minutes after birth. If a doe rejects a fawn, colostrum replacers can be used to provide antibodies, but nothing is better than the doe’s colostrum. Fawns with greater immunoglobulins (antibodies) at 24 hours after birth had greater CONTINUED ON PAGE 38» 36
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» FAWN NUTRITION
« CONTINUED FROM PAGE 36
survivability than those that did not survive. Lack of colostrum consumption and failure of passive immunity in deer and livestock can increase the newborn animal’s susceptibility to septicemia, diarrhea, other disease, and increase risk of mortality. As one would expect, does with a 20% decrease in food resources during lactation had fawns with lower growth rates during the first 30 days post-fawning. If the doe produces inadequate amounts of milk or rejects the fawn, an appropriate milk replacer should be used to feed the fawn. In a breeding facility, this is also a good time to consult the herd veterinarian to ensure appropriate vaccines and other health procedures are completed. Fawns begin nibbling on dry feed starting at two weeks of age. In deer breeding facilities, this is a good time to provide a fawn diet in a creep area that is only accessible to fawns. The fawn diet should be palatable to attract the fawns
to the creep area and be kept fresh. It is also ideal for the fawn diet to be similar to the adult diet to allow an easy transition at weaning time. The goal is to provide supplemental energy and nutrients to the fawn in excess to what the doe can provide through the milk. The creep area should be an area out of the weather with clean dry bedding and, fresh-clean water. The fawn diet should be fed through weaning and slowly replaced with the adult diet during the weaning process. By ensuring does are fed appropriately during late gestation and lactation and fawns receive colostrum and get started on dry feed, fawns will have a successful beginning. Providing optimal nutrition early in life will support fawn growth, body condition and fawns will more likely express their genetic potential for antler growth, reproduction and have productive futures. u
Dr. Schlegel received his B.S. in Animal Production from The Pennsylvania State University and a M.S. and Ph.D. in Ruminant Nutrition from the Department of Animal Science at Michigan State University. He completed a joint postdoctoral research fellowship with the University of Florida’s Department of Animal Science and Disney’s Animal Kingdom Animal Programs in exotic animal nutrition. Dr. Schlegel is a Certified Professional Animal Scientist and a Diplomat of the American College of Animal Sciences – Nutrition Discipline. He taught in the Department of Animal Science as an Assistant Professor at Delaware Valley University (Doylestown, Pennsylvania). Dr. Schlegel was the Director of Nutritional Services at San Diego Zoo Global from 2005 to 2015. Dr. Schlegel joined Purina Animal Nutrition as a Senior Nutritionist in November of 2015 and is part of the Technical Innovation team focusing on wildlife and small ruminants. Dr. Schlegel is a member of the Comparative Nutrition Society, American Society of Animal Science, and American Registry of Professional Animal Scientists. He has been a section editor for the Journal of Zoo and Wildlife Medicine, and has authored numerous peer-reviewed articles, book chapters, proceedings, abstracts and research reports. 38
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2019-2020 BR E D DOE S Doe bred to Brain Freeze •Knockout/G2/K t/G /K K Kingpin/K Kristie
Does bred to Bu uckle Up •Freightliner/2 20-28/PK 8 24 •20-28/Amb ber
Does bred to Gunslinger •Triple Crown /Overnight E xpress/ Hardcore/ W 7 2 (E scalad des Womb)
D oe s bre ed d to Uno An A c ho •Gunslinger/ er Overnight E xpress/ Gladiator/ Kingpin/ P625 • G2 /838 (P h hen noms Dam) •G2 / Texas Rang n er/G2 sister •Af tershock/ k /Monarch Supreme/ Hardcore/ W 72 (E scalad a es Womb)
Does bred to Black kJack k •Monarch 1/ 20 -28/ High hroller/ W 7 2 (E scalades Womb) •Higharchy/Monarch Sup u reme/ Hard d co r e / W 7 2 (E scalades Womb) •Triple Crown/Jesse ss James/ E scalade/ W 7 2 (E scalade a s Womb) •High Heat / Bambi 7 27/ Bul B lwi w nkle •Unforgiven/ Triple Crown/Jesse s James / Pk003 0 6
Doe bred to Veg gas •Gunslinger/Masterpiece/Ranger/Green8
D o e s b r e d t o L e ga c y •Triple Crown/ Wallkin Tall /Gladiator/ B20 •Higharchy/ E xpress/Monarcch 1/ Yankee •Midnigh g t E x p r e s s /M o n a r c h 1 f u l l s i s t e r •Freightlinerr/ 20 -28/ PK 24 •20 -28/A / mb er
Does bred to Horsepower •Unforgiven/ Triple Crown/Jessse James/ Pk003 0 6 •Freeze Frame/ Triple Crown/ Yellow Rose (E xpress/A 225 kid rock s dam)
D o e s b r e d to M ajo r L eague •Triple Crown/ n E xpress/ Brandi •E xpress/G2 full sister
D o e s b r e d to B lue c hi p •Overnight E xpress/ Hammerdown sister •Unforgiven/ Freeze Frame/ E xpress/ Ranger/ Green 8
Wa t c h o u t f o r t h e s e P o w e r D o e s c o m i n g s o o n •Bluechip/ Horsepower/Shadow/ L auren •Holly wood/ Unforgiven/Overnight E xpress/Monarch Classic sister •WD -40/ Horsepower/Sudden E xpress/ Hardcore •Enterprise/Masterpiece/Maxin/Shadow/ Rolex sisster
M at t L o essi n | O w n e r | m lo essi n @ v ar a r an c h . c o m | 9 79 -7 32- 0 2 3 4
Trey S t a fff | R an c h M an age r | t st a fff@ @ v a r a r an c h .c o m | 9 79 -7 3 3 - 6 3 0 4
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» CERVID HEALTH
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Fusobacterium Necrophorum By Cervid Solutions
with contaminated soil it can result in infection. Overcrowding and stress can propagate diseases caused by this bacteria. • Changes in the rumen environment, such as grain overload or sudden changes in feed can damage the stomach lining and provide a portal of entry for Fusobacterium where bacteria may pass to the liver causing hepatic necrobacillosis (death of liver tissue), or move on to other organs. What symptoms are typically seen in Cervid? • Fusobacterium is transmissible through contaminated • Trench Mouth (commonly referred to as Lumpy Jaw) feed or water troughs from an infected animal. • Foot rot • Fusobacterium minimally affects more than one deer • Respiratory problems, Pneumonia at a time but can affect several fawns. • Abscesses • With the case of foot rot injury to the interdigital skin • In rare cases it can be found in Uterine Infections provides a portal of entry for infection. Foot rot is also and Mastitis transmissible to healthy animals; it requires a warm, moist environment. Under these conditions, the interdigital How is it spread? • The organisms gain entry into the body through cuts stratum corneum becomes macerated (moist softened tissue and abrasions to the skin or mucous membranes. In the in a state of deterioration); filaments of F. necrophorum mouth many outside influences could cause abrasions, such invade the superficial epidermis and induce interdigital as coarse feed or metal objects along with unevenly worn or dermatitis. new erupting teeth which can penetrate or damage the oral lining and create points of entry. Fusobacterium survives CONTINUED ON NEXT PAGE» well in wet soil and when mouth abrasions make contact • “Anaerobic” gram negative bacteria. Anaerobic simply means it likes to grow in places with little oxygen. • Causes Necrobacillosis, the definition of necrobaccillosis is any disease marked by necrotic (death of cell tissue) areas in which bacillus (rod shaped or cylindrical bacterium) is found.
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» CERVID HEALTH
« CONTINUED FROM PAGE 36
Foot rot • Lameness is seen when the feet and associated joints geography are involved. Swelling between the toes is the first sign seen, • F. necrophorum occurs throughout the world. followed by localized tissue death, spreading to the joints • New Zealand and North American deer farms suffer and bones in more advanced cases. In general pain, severe significant losses from foot rot and necrotic stomatitis lameness, fever, anorexia, loss of condition and reduced milk (tongue and throat abscesses) from the diseases. production can be major signs of the disease. Signs and Symptoms Lumpy Jaw • Lumpy jaw also appears as a swollen jaw or cheek. It is the result of an infection of the jawbone. These lumps are immovable hard swellings of the bones, usually at the level of the central molar teeth. The swellings develop slowly and may take months to reach the size of a golf ball. They consist of honeycombed masses of bone filled with yellow pus that comprise the lumpy jaw. The swellings may become very large and discharge small amounts of sticky pus containing gritty yellow granules. 44
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respiratory problems, pneumonia • In the throat, necrotic laryngitis will show itself as loud wheezing. Some dead tissue and bacteria may be sucked into the lungs causing abscess formation and pneumonia. uterine infections • There is a lack of research on uterine infections in the cervid community, but in a study of small flocks of sheep in Denmark, 4 of 24 ewes were found to have aborted from F. necrophorum. There was necrosis of the cotyledons of the placenta, which is where the maternal fetal exchange takes place. nADeFA.org
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• F. necrophorum can also cause metritis (inflammation of the uterine wall) and endometritis (inflammation of the endometrium). A fetid, watery uterine discharge is characteristic of this condition in cows but may be conspicuous in other species.
Statistics • In a study from 2003 on mortality rates of captive PA deer, bronchopneumonia was the most common cause of death. The majority of cases (64.1%) of bronchopneumonia occurred in deer 2 years of age or less with 46.2% of the cases seen in animals 1 year or less. F. necrophorum was one of Mastitis • There are 4 different clinical types of mastitis but the most commonly isolated bacterial respiratory pathogens generally the signs are swelling, heat, pain and abnormal from the affected lungs, accounting for 27.8% of the cases of secretion in the gland, accompanied by fever and other signs bronchopneumonia. of a systemic disturbance such as marked depression, rapid weak pulse, sunken eyes, weakness, and complete anorexia. points to remember Disease Management • Proper management is important to minimize disease and reduce the contagiousness of the bacteria. Keeping deer in a clean, minimal stress environment and not overcrowding them will decrease the spread of the disease. Stress such as heat, cold, overcrowding or poor nutrition predispose to infection. • According to the USDA approximately 1/3 of all deer and elk operations vaccinate against fusobacterium Only 17.8% of deer operations vaccinate against F bacterium. • Autogenous vaccines are an option in the prevention of these bacteria.
• Mostly seen as Trench Mouth, Foot Rot and Pneumonia. • Fusobacterium • can gain entry to the body through cuts or abrasions to the skin. • can enter through the lining of the stomach if there is abrupt change in the rumen environment. • is transmissible through contaminated feed or water. • can be passed from animal to animal as foot rot. • Vaccination is key to prevention, Pneu-Vac-2 covers Fusobacterium Necrophorum. u
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» NADR REGISTRY NEWS
Have You Converted Your Herd To SnpS?
• • • • • • • • •
Determine what animals you are and will be breeding with in the future All animals tested since 2017, have been converted to SNPs How to determine if an animal has been tested utilizing SNP technology You can do this by logging into NADR Interactive and selecting the animal. If it has been tested, it will say “Complete”, otherwise it will say “None” There is also a “SNP Profile” column on the grid with all animals in your inventory Any future fawns submitted will only utilize the SNP technology for testing as long as the Sire and Dam have been tested with the SNP technology When you work your deer this year, pull your buck and doe samples as soon as convenient for you. Call the NADR Office before you send them and we will work on a plan to test them utilizing SNPs as fast as possible Once converted, the price per animal will drop to $55
Go to the link below to view NADR’s new video. https://www.youtube.com/watch?v=X9mcoEMtacA
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prepare For Fawning Season As we prepare for the upcoming fawning season, NADR wants to make you aware of some exciting technology improvements to assist in your breeding efforts. Tissue Sample Collection Method - We all know that most of the industry utilizes hair samples. This is an extremely laborious way of collecting samples and not to mention increases contamination between collection of different animals due to the hair blowing and sticking to everything it comes in contact with. The solution is a tissue sample method known as TSU Tubes utilizing the All-Flex Tissue Punch. This method has been used by our laboratory, GeneSeek for many years in other animal genetic testing. These unused devices are said to have an indefinite shelf-life but since they contain some cell lysis material in the buffer, we would recommend to only keep for two years. Once the devices are used, they will remain stable for at least 1 year at room temperature. Our lab will freeze Spring 2020
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the remaining tissue/buffer once the DNA has been extracted. This method will be more efficient as well as eliminate the mess associated with hair samples. The tubes have a unique number and barcode so they will eliminate confusion with any other animal. With a minimal investment of $50 for the applicator, you can be utilizing the new sample method in a few days upon placing your order. These tubes can be purchased from GeneSeek for $2.10 plus a small handling charge. The applicator and the tubes can be purchased at https://order.igenity.com/ NADR will reduce your parentage costs by $3 per tube submitted to the registry for testing. This will ensure you are not out of pocket any monies when utilizing this technology. For further information on how this sampling method works, take a look @ this video. h tt p s : / / w w w.yo u t u b e.c o m / w atc h ? v = K X E 5 p LTRJU&feature=youtu.be North American Deer Farmer
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» NADR REGISTRY NEWS
Snps vs. STrs Who Cares and Why the Change?
Almost 150 breeders have converted their herds to SNPs. With that said, I know there continues to be some confusion regarding SNP Technology and I wanted to shed some light on this subject and effectively communicate answers and solutions to your questions and comments which are shown below:
will be utilized side by side with our old technology, STRs, until each customer is fully transitioned over. At that time, we will only utilize SNP technology for parentage testing. The advantages will be: 1. Lower costs 2. Easier to interpret data and results
1. Why did we switch to SNPS? 2. If this SNPs technology is better; then how can we switch sooner rather than later? 3. Why does NADR need to call me in order to determine parentage? 4. Why can’t NADR just take my samples and plug the DNA into a database and get an answer?
Lineages continue to become tighter making parentage more challenging than ever before. NADR’s Board had enough forethought to predict this problem would not get better but only get worse if we didn’t do something to create a DNA test that would make parentage easier. That is where SNP technology was introduced. With the continuation of the tight lineages, the STR technology cannot be sustained NADR started using SNP technology for parentage or take the deer industry into the future and produce beginning in 2017. This new technology is called SNPs and parentage results that can be trusted. Most breeders are 50
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experiencing some of these problems now and they will 1. Determine what animals you are and will be continue to be magnified every year until each breeder is breeding with in the future fully converted. a. All animals tested since 2017, have been This isn’t something new in the animal testing world. To converted to SNPs put things in perspective, the cattle industry as well as other 2. How to determine if an animal has been tested industries such as swine utilize thousands of SNP markers utilizing SNP technology to test their animals, compared to our 18 STR markers and a. You can do this by logging into NADR Interactive 400 plus SNP markers we use for deer. This was done and selecting the animal. If it has been tested, it will primarily to manage and understand the genetics of their say “Complete”, otherwise it will say “None” animals in which the industry was tightly bred as well. b. There is also a “SNP Profile” column on the grid NADR has and will continue to add markers to our with all animals in your inventory parentage assay to ensure what is happening now will not 3. Any future fawns submitted will only utilize the SNP happen in the future. technology for testing as long as the Sire and Dam SNP technology will be more cost effective for NADR have been tested with the SNP technology and the breeder. It is more amenable to robotics which reduces human technical errors and improves efficiencies 4. When you work your deer this year, pull your buck which naturally bring costs down. It is the gold standard for and doe samples as soon as convenient for you. genetic improvement of livestock breeding and is essential 5. Call the NADR Office before you send them and we for the future of our industry. will work on a plan to test them utilizing SNPs as fast Just to be clear, parentage is still being performed as possible utilizing both STR and SNP technology until such time all 6. Once converted, the price per animal will animals on your ranch utilized for breeding have been tested drop to $55 with the SNP technology. Any animal tested since January We appreciate your feedback as well as constructive 2017 has been tested using SNP technology. If you are breeding with any animals that were tested prior to 2017, criticism and will continue to look for ways to use they must be converted to the new SNP technology in order technology to make improvements for the deer industry. u for your herd to be fully transitioned. Go to the link below to view NADR’s new video. How can you speed up your own transition to SNPs? https://www.youtube.com/watch?v=X9mcoEMtacA You can proactively do the following:
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SHOTGUN
EXCAL
NEEDLES
GEORGE POW WER DAM
CRAZY HORSE
POWER DAM
BLACK HHAWK
C CHIEF CHEYENNE
SHOTGUN RED 5
8SS BLUE
YELLLOW 88
A Ceda ar Breaks Ra anch & Misso ouri Valley M Muleys PA ARTNERSHIIP
» CWD RESEARCH
Have You Heard of
Humic Acid? It seems every year there is a new product or additive that falls under what I call the “silver bullet” category. Most of these miracle cures are typically marketed toward helping with CWD, EHD or improving antler production beyond imagination. In my experience, most of these silver bullets get debunked over time due to not showing enough response to warrant the cost of the “silver”. These additives are usually nonmedicated nutraceutical or natural additives that have often shown promise in other species when it comes to nutritional performance. Tangents are typically drawn to whitetail and responses warranted are often not supported by any significant data though. With that being said, when I was first approached about possibly finding a source of Humic Acid (HA) to put in 56
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deer feed, you can say I was skeptical at best and assumed it was simply the next “silver bullet”. To my surprise, the more I looked into HA and talked to breeders that have used it, the more intrigued I have become when it comes to this additive. HA is nothing new, it has been around for a long time and used throughout the agriculture industry. HA has been well documented in nutritional trials particularly on the beef and dairy side of the fence and shown to improve microbial function in the rumen and digestive tract of cattle. This in turn positively influences the productivity and the quality of their production. What really makes HA appealing is that natural organic additives are typically a better alternative to antibiotics. Quantifying performance of these nADeFA.org
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types of “natural” additives is where it gets fuzzy though when it comes to whitetail deer. What we know: HA is a long-chain molecule high in molecular weight, dark brown and soluble in an alkali solution [Stevenson, 1982]. HA is used in ruminant diets because it improves microbial production leading to improved energy provided to the animal. This possibly leads to other health benefits like improved digestion and feed utilization, which should also improve growth, and immune system function. Humic acid also has absorptive and detoxifying properties [Trckova et al. 2005]. Now what we don’t know: Due to the detoxifying properties of HA, some deer breeders believe that it may work to denature the CWD prion when provided in certain concentrations in the diet of whitetail deer. While it is my sincere hope that this is ultimately true, I have seen no significant data to prove that this response is guaranteed outside of a petri dish. Other downstream responses noted by breeders that have used this additive are improved feed utilization, and immune response improvement. Most notably mentioned were decreased incidence of respiratory and other bacterial complications that are typically common with whitetail deer in captivity. Other response noted was improved body condition and overall appearance of the deer. Based on what we know about this additive, the body condition and appearance piece holds true with what has been proven as major improvements in microbial production and function in ruminants typically exhibit these types of positive feed performance responses. The source of HA has also been a grey area as different sources will have different concentrations of HA, so a standard inclusion really depends on the concentration of HA which will most likely vary from one batch to the next on sources that are not approved feed ingredients. Although not mentioned, I believe different sources could also affect palatability particularly with whitetail deer. In my experience subtle changes even with micro ingredients can knock deer off feed and ingredient consistency is crucial when it comes to maintaining intake. Some breeders adding this to their ration are using sources that are not approved animal feed
ingredients which I do not recommend. There are commercially approved feed ingredients that have concentrated levels of HA that I recommend be used in order to see any results replicated and not see antagonistic issues with contaminants or other minerals. So what do I recommend? The upside is too good not to try it in my opinion. We know we should see a positive microbial response which would translate to improved feed efficiency, nitrogen retention, starch digestibility and hopefully immunity. Record Rack has identified a proprietary additive that is a concentrated source of HA and have added it to our Stress Pack at the recommended inclusion rate. The Stress Pack is currently available in Texas to add to any of our rations in minimum run bulk or bag quantities for breeders that want HA in their feed. Its important to remember that this pack isn’t just HA, it also provides other probiotics and improves levels and absorption of Vit A,C,D and E all at a relatively low price tag. Will this cure CWD and eliminate all bacterial challenges we see in deer pens? Most likely not, as there is no way to ensure that outside of putting deer in a bubble. Could it mitigate bacterial challenges and possibly have CWD or EHD implications? Again I sincerely hope this is the case, but further trials and more deer on feed will be needed tell the tale. I do believe this additive is a viable additive that could improve feed performance if more is warranted or if you are really pushing the energy threshold in your pens. Further research will definitely be required to uncover any particular epizootic guarantees and believe base ration formulation, management practices, stress levels and pen dynamics will all play a major role in response seen. You have to love being where we are in the industry nutritionally as we learn something big every year it seems. In my opinion, if you are not working with a nutritionist to improve your nutritional program every year, you are most likely already behind. Is HA the next big thing? We will see. u Thanks, Bobby Deeds Wildlife Specialist record rack
Stevenson F.J., 1982 – Humus Chemistry: Genesis, Composition, Reactions. 1st edn. Wiley. New York, USA Trckova M., Matlova L., Hudcova H., Faldyna M., Zraly Z., Dvorska L., Beran V., Pavl ýk I., 2005 – Peat as a feed supplement for animals: a review. Vet. Med. – Czech, 50(8): 361–377.
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» NADEFA WORKS FOR YOU
Wow! That’s how much money NADeFA and the CLF have put directly into protecting and promoting this Industry in the last 11 years. The total does NOT include ANY money spent on operating expenses (salaries, office expense, travel, advertising, etc.). This total was all spent on You and Your industry! Here is the breakdown: Funds given Directly to States for their use
$348,984
Legal Fees for State issues
$216,150
Lobbying • Capitol Hill Consulting Group • Lobbyists or state issues
$876,514 $793,024 $83,490
research • Research Programs Include: Chembio, Bio Tracking, WOW Foundation, Cornell University, Texas A&M, Texas Tech, Kansas State University, Midwestern University for Issues such as CWD, EHD, TB, Gene Research, Breeding, Pregnancy research and Reindeer research.
$382,557
Education and Educational materials • Deer in School Program • Educational Materials • Economic Impact Study • Venison Competition Scholarship • PR Materials • New Deer Farmers Seminar • College Scholarships • Intern Program
$383,438
Television • Keith Warren • Conquest 200 uSDA
$70,960 $82,461 $15,000 $22,900 $87,394 $34,388 $24,000 $46,335 $589,500 $527,200 $62,300 $26,586
If you are asked exactly what it is that NADeFA does for the industry, you now have the answer. NADeFA dedicates every day to making the cervid industry a strong viable business that will be in existence for years to come. Encourage everyone you know to become a member of the organization that works for its members and the entire industry
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NEW MEMBERS Dustin Anderson Bayou Bugs and Bucks
Kirsten Krause nFp
Larry pritchard The Monarchs of Keo
gene Ballard Big Horn Whitetails
Frank Landry Texas Deer Factory
Steve ramberger Emery S. ramberger Farms
James D Beechy Double E Whitetails
Brittney Lepley Camelot ridge resort
David redd rrgo
Mark Behme Behme's Whitetails
Blaine McClung Whitetail Heaven
Wendy Scanlon Deer Breeders gazette
David Caisse game Changer Whitetails
Devon Milligan rockin' M Farms
patrick Stanley Crooked Creek Farms
Juan M Campos Krauer university of FloridaÂ
robby Mills Mills Whitetails
Jason Stefanowicz Awesome Whitetails
Micah greathouse Kaiser Creek ranch
Joshua neal neal plantation Whitetails
Larry Wagler Skyline Whitetails
Steven Haines ridge runner Whitetails, LLC
Kurt nielsen Vital Way Whitetails
Josh Wagner JW Whitetails
Brad Hensel Hensel Whitetails
Stephen patterson Thornhill Whitetails
Samuel Whitaker Whitaker Whitetail Farms
Mark Hollenbeck Sunrise ranch LLC
Eric paul
Edwin M Yoder Easy Whitetails
Mark pettus Soggy Bottom Lodge
Todd Hosch Hosch-A-Bye Whitetails
Wilbur W Yoder Stutz Creek Whitetails
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CAPITOL HILL REPORT
D.C. Is Physically Closed But Business Is Moving Forward Offices on Capitol Hill are physically closed, Washington is as busy as ever. While our normal legislative calendar is out of sync, there has been plenty happening in both Congress and the Administration. Congress has enacted a number of measures to help the economy since COVID-19 brought much of the country to a standstill. Provisions to help agriculture producers have been some of the slowest to reach the intended target, and have been uneven in their effectiveness. Most recently funds to help livestock producers was limited to major meat species that have seen their market prices drop. We successfully added cervids to USDA’s list of livestock in the past, but this targeted application has left us out. We are engaged with lawmakers and USDA to determine how we can rectify this hopefully in this application, but for certain in any future packages. There has been a renewed interest in Washington in shutting down illegal animal trading that fuel “wet markets” and similar operations such as the ones in China that may have help spread the COVID virus. Some are proposing eliminating any movement of wildlife in any circumstance. This would be a tremendous coup for animal rights activists. We are engaged to educate Congress and the Administration on how this could inadvertently hurt cervid farmers, as well 64
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as those that raise exotics, including critically endangered species that the industry has helped preserve in the face of extinction. This is an ongoing concern that we expect to be at the forefront for the foreseeable future. Amidst the chaos, there is some normality returning to Washington. The appropriations process, which establishes funding for the federal government each year, is starting back up. While it is starting later than planned, we expect it to move more or less as normal. This is good for our efforts to increase funding for research into CWD and indemnity payments for producers who have to depopulate their herds. We missed seeing you all in Indiana for the conference or here in Washington for our annual fly in, and can’t wait to see you all. We hope things are somewhat back to normal before the rut hits and you have hunters in camp and active markets. Enjoy your social distancing on the farm and we’ll be in Washington protecting the industry for generations to come. u Sincerely, Stratton Edwards and Jack Victory Capitol Hill Consulting
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SPRING 2020
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» FAWN HEALTH
Killer Fawn Disease one Farmer's report on an important Discovery by Cleve Tedford, Mule Face Farms
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"Sphaerophorus Necrophorus." With those two, cryptic words, NADeFA past president Dr. Raleigh Buckmaster identified the organism responsible for the high level of fawn mortality in our Fallow Deer herd. It was early August and Bette and I were in Washington for the House hearings on Tb in cervidae. Just prior to the hearing and Dr. Buckmaster’s testimony on the NADeFA position on Tb indemnification, we were talking Deer Farming over lunch. After I had described the circumstances surrounding the sudden death of up to 20% of our recent fawn crop, Raleigh named the culprit. This was subsequently confirmed in necropsy reports from the University of Tennessee, College of Veterinary Medicine. Throughout the rest of this report, Sphaerophorus Necrophorus will be referred to by the more commonly used description, Fusobacterium necrophorum or simply, Fusobacterium. The circumstances of our high level of fawn mortality were as follows: 1. After an uneventful fawning and lying out period of about one week, the fawns began to move about, accompanying the does to the feeders. 2. Within a period that varied from one to six weeks after birth, a fawn that had been observed healthy and moving about the field and feeders one day would be discovered lying off by itself the next. 3. When the group was called to 'the feeders, the afflicted animal would remain behind in the field. 4. Within 12 to 48 hours, the fawn which was observed becoming weaker and refusing to nurse, died. 5. Two fawns which were removed from the field as
soon as they were weak enough to capture subsequently died despite tube feeding and injections of antibiotics. 6. Post mortem examinations by our local veterinarian revealed an ugly abscess in the liver, clinically described as a "10 em. cavity filled with thick yellow flocculent exudate" (pus). The final necropsy reports from the University of Tennessee, which we received several weeks after the enlightening discussion in Washington, yielded the following additional observations and conclusions: 1. Ulcers in the rumen, which in at least one fawn were determined to be the point of entry of Fusobacterium. 2. Ulcers of the mouth and tongue, which were determined to be the point of entry of the Fusobacterium. 3. Severe necrotic ulcers of the skin between the toes of at least one fawn, diagnosed as another possible point of entry. 4. Diagnoses: (a) Liver: Severe chronic necrotizing hepatitis due to Fusobacterium necrophorum. (b) Tongue: Severe chronic necrotizing lingitis with filamentous Gram negative bacteria Fusobacterium necrophorum. (c) Rumen: Severe necrotizing rumenitis with intralesional filamentous bacteria Fusobacterium necrophorum. (d) Distal limbs: Severe acute ulcerative dermatitis with myiasis. 5. Cause of Death: Polysystemic Fusobacteriosis. CONTINUED ON NEXT PAGE»
Editor’s note: Cleve Tedford, who was scheduled to make a presentation: “31 Years Farming Fallow Deer” at our Annual Conference in French Lick, has agreed to contribute information on lessons learned the hard way. A former Director of NADeFA and the Cervid Livestock Foundation, Cleve has represented North American Deer Farmers and made presentations at international meetings in Australia, New Zealand, England, and the Czech Republic. Author’s note: The following article appeared 27 years ago in our nADeFA magazine and other alternative livestock publications both in America and overseas. At that time over 90% of NADeFA members were farming Red and Fallow Deer with around 10% raising whitetail. By 2011 those figures were reversed, At our Annual Conference in Nashville that year, in a breakout session on Herd Health attended by over a hundred whitetail breeders, the most urgent questions from the audience concerned the sudden death of fawns. I was given the opportunity to speak and related my earlier experience with Fallow Deer. Apparently, few breeders of whitetail were aware of Fusogard in 2011, and lots of notes were taken. When the following article was published, Fallow breeders in America weren’t aware either, but within a month we had located Fusogard and were vaccinating our does. PROBLEM SOLVED! Spring 2020
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Âť FAWN HEALTH
ÂŤ CONTINUED FROM PREVIOUS PAGE
By the middle of the summer, the deadly kiss of Fusobacterium had turned my daily walks through the fawning pastures, which I formerly counted the most enjoyable and satisfying experience of deer farming, into a dreaded chore. Observing and identifying the problem was emotionally draining, to say the least. Before continuing, I would like to assure the faint of heart that this is not another Mycobacterium bovis. Hang on, if you will, and don't let your anxiety cause you to nervously skim the rest of this report. You won't mind the term "whole herd depopulation" here. Determining the cause, cure and a prevention program for this scourge which had wiped out nearly 20% of our fawn crop became the number one priority of our whole farming operation. Early on in dealing with our first few fawn deaths, I was somewhat diverted in my search for a cause and cure
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by a fawn which exhibited all the symptoms of Navel ill, a common problem in other ungulates. Those symptoms were severe infection of the navel and adjacent tissue, swollen joints, weakness, immobilization, etc. We kept this fawn alive for over a week with massive doses of antibiotics and tube feeding; however, as its condition grew worse, our only humane option was to euthanize it. Subsequent post mortem revealed the ugly abscess in the liver, typical of Fusobacteriosis: At this point, I determined in my own mind that the mode of entry for the organism was through the navel cord for the following reasons: 1. We had unusually wet weather during our fawning season of June/July and new born fawns were often observed lying in muddy areas around the edges and corners of our fawning pastures. 2. A spring branch ran through one of our fawning pastures, and I often discovered to my horror that the fawns nADeFA.org
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were hiding out by lying in wet areas adjacent to the stream. 3. Both of the conditions cited above prevent the navel cord from drying out, providing an open conduit for the Fusobacterium. From what I knew about Navel ill in other livestock, it could normally be prevented by dipping the navel cord in iodine at birth. Although usually not an easy thing to do with fawns, I said, "so what!" I was going to do it anyway, and next year my weaning rates would be back up to 90%. Unfortunately, as the final necropsy reports began coming in from the University of Tennessee, I discovered that the point of entry in the majority of cases was through the mouth. As this began to sink in, I realized that the few small patches of blackberry briars and multiflora rose that I had left along the spring branch "for the deer to clean up" were causing the small cuts in the mouths of the fawns that were providing entry to Fusobacterium. Early in June, when first confronted with the high fawn mortality rate, I had immediately assumed that the cause was abandonment/starvation. I arrived at this convenient conclusion after my own rudimentary post mortem examinations revealed that the victims had empty stomachs. Also, there was no diarrhea or other symptoms usually associated with the death of young livestock. Flush with this bit of "vital" information, I had faxed friends at AgResearch (formerly MAF) in New Zealand explaining my problem and inquiring as to their experience with mass outbreaks of abandonment/ starvation. They were very sympathetic and helpful, providing an analysis of the problem within the parameters I had set…abandonment/starvation. I was very much off the track. In early August, when with the words "Sphaerophorus Necrophorus," Raleigh Buckmaster cracked the code to this whole messy mystery, I got back on the FAX to the deer experts in New Zealand and quickly learned from them that, yes, Fusobacterium regularly wreaked its havoc among their deer herds resulting in mortality rates as high as 30%. There it is referred to as Navel ill, Wooden Tongue and Lumpy Jaw in reference to some of the visible outward symptoms. Spring 2020
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[Note that these terms are used by US cattlemen to refer to syndromes in cattle caused by other soil saphrophytes which occur under similar conditions. Fusiform is US cattle causes a symptom known as "Foot Rot." +ED.] The good news was that its is possible to vaccinate against the ravages of Fusobacterium through the use of a product called FRA Vaccine produced in Australia. Fusobacterium is the causative agent in foot rot in cattle; and although FRA Vaccine was developed for the cattle industry, its application in deer farming was proving most effective. I have since learned that FRA Vaccine has become hard, if not impossible, to obtain. Ironically, about the same time I learned of the vaccine, I saw an advertisement in The Deer Farmer (New Zealand) advertising Fallow deer. Included in the ad was the statement that the animals were FRA vaccinated. The ad was placed by Ernie and Leonne Hazelhurst who have contributed as much to the development and promotion of the farming of Fallow deer as anyone else in the world. Ernie is past president of the New Zealand Fallow Deer Society and is currently editor of their newsletter. He is also the work horse behind the scenes of the World Forum on Fallow Deer Farming to be held in Australia March 10-13, 1993. Finding the ad led to more faxes and correspondence with Ernie who generously provided an overview of the status of Fusobacterium disease in New Zealand and Australia. He also stated that "It is the cruelest disease I have ever witnessed in any livestock. Once fawns have contracted it, they cannot be saved; but, yet, it can be controlled." Controlling it by prevention is still the number one priority on our deer farm. Raleigh Buckmaster sadly told me that to his knowledge there was no vaccine commercially available in the US. After several weeks of research and phone calls, I understood what he was talking about: NO VACCINE. Toward the end of August, I had the occasion to talk with NADeFA Vice President, Scott Petty, Jr., another gentleman like Ernie Hazelhurst, who has made enormous contributions to the development and promotion of deer farming. I explained the problem I was having with Fusobacterium and my sad discovery that there was no vaccine available in this country. CONTINUED ON NEXT PAGE» North American Deer Farmer
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» FAWN HEALTH « CONTINUED FROM PREVIOUS PAGE
A few weeks later I received from Scott a copy of a letter he had received from Dr. Bruce Burton, Health Committee Chairman of the British Columbia Fallow Deer Association. Dr. Burton's letter filled in many of the gaps in my knowledge of what the Canadians call "Killer Fawn" disease. One conclusion I drew from his information was that you can run, but you can't hide from Fusobacterium. His letter included mention of outbreaks in New Zealand, Australia and Canada, as well as cases involving mule deer and whitetail deer on the Canadian prairies and even reindeer in northern British Columbia. The organism seems to be lying in the soil everywhere just waiting for the right conditions to begin it's devastation. The unusual amount of rain we had last summer during our fawning season seems to have provided the right conditions, aided and abetted by the spring branch, briars and multiflora rose in my fawning pastures. At last, we do have hope of obtaining a vaccine that will allow us to again enjoy our walks through the fawning pastures in June. For several months now, our friends at the University of Tennessee have been working with a laboratory toward the development of an autogenous vaccine. We are confident that by next June, all the necessary preparations will be made to overcome "Killer Fawn" disease. These include: • replanting all bare areas in our fawning pastures
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• maintaining areas of tall, rank grass in dry, central areas of our fawning pastures for the fawns to lie out in • removing all briars, rose vines, sticks and any other materials on which the fawns might tend to gnaw • vaccinating all does at least twice prior to fawning • preventing access by fawns to areas with streams • disinfecting navel cords with iodine at birth • spraying disinfectant on bare, muddy areas that may develop in fawning pastures. At this point I would like to say that without the help of Dr. Raleigh Buckmaster, Scott Petty, Jr., Dr. Geoff Asher, Martin Langridge, Dr. Bruce Burton and Ernie Hazelhurst, we would still have only the most rudimentary knowledge of the problems associated with Fusobacterium. This information might have taken years to acquire were it not for the contacts and relationships that have been established through our membership in the North American Deer Farmers Association. If it is all new to you and can help you save the life of even one fawn, then you have been more than compensated for the cost of your NADeFA dues. If you are not a member, I encourage you to join. If you are already a member, give some serious consideration to contributing additional financial support and time to your association. You will find that the dividends you receive from your investment will be those of the best and cheapest means of improving the profitability of your deer farming operation. u
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» CHeRI
WHAT IS THE CERVIDAE HEALTH RESEARCH INITIATIVE? Samantha M. Wisely, Professor of Wildlife Ecology and Director of the Cervidae Health Research Initiative, Institute of Food and Agricultural Sciences, University of Florida
The Cervidae Health Research Initiative, CHeRI, is a university/industry partnership between cervid farmers of Florida and the University of Florida. Using the best available science, CHeRI seeks to find solutions to the industry’s greatest problems to herd health and production. Funding for this initiative comes from the Florida State Legislature as a result of lobbying efforts from the deer farming industry in Florida. After devastating herd losses from epizootic hemorrhagic disease in 2012, Florida deer farmers requested help from the legislature and University of Florida. The result was the introduction of CHeRI in 2015. THE WorK oF uF/iFAS CHEri Defining the Viruses that Cause Hemorrhagic Disease – We listened to what deer farmers had to say, and it is becoming clear that hemorrhagic disease was the number one threat to economic sustainability in Florida. Our first priority was to provide a diagnostic service to deer farmers so that everyone could truly understand what was killing deer in Florida. This effort helps deer farmers support sick animals and take preventative measures; it helps CHeRI scientists to develop effective vaccines against viruses. Results from the diagnostic services provided by CHeRI and the samples provided by deer farmers have changed the way we view this disease. While epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) remain the predominant viruses that kill deer in Florida, we have discovered 5 additional viruses that are closely related and likely result in hemorrhagic disease in deer. CHeRI has now partnered with several vaccine makers to produce vaccines against these viruses.
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inTEgraTED pEST MAnAgEMEnT oF no-SEE-uMS The viruses that cause hemorrhagic disease are transmitted to deer by insects. Understanding which species of insects carry the viruses is critically important to developing control and management plans. CHeRI entomologists have discovered which no-see-um insects carry EHDV and BTV in Florida, but we still need to understand which insects carry the other viruses that cause hemorrhagic disease. The CHeRI No-See-Um Identification Services Project provides farmers with a farm-specific profile of disease causing insects on their farms. The data generated from this project helps scientists understand the distribution of these insects and identify which ones cause disease. pAraSiTE rESiSTAnCE To TrEATMEnT Farmed deer can carry worms, helminths and protozoans that reduce the growth potential of deer. Most farmers treat deer for worms regularly, but are these treatments effective? CHeRI researchers are investigating drug resistance in gut parasites of deer. CuriouS ABouT WHAT ELSE CHEri DoES? The UF IFAS Cervidae Health Research Initiative conducts research on problems affecting farmed deer, provides free disease diagnostic services to Florida deer farmers, and is training the next generation of deer vets and farm managers. To learn more about the science and meet the scientists of CHeRI, visit our website (https://wec.ifas.ufl.edu/cheri/) where you can find publications, videos and fact sheets about deer health. u nADeFA.org
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TREASURER REPORT
Membership Is Key To Our Association... Help Bring New Members Last issue I commented on the wacky weather here in New England. Well, the unpredictability has continued. It snowed two weeks ago and now in the last week of May it’s 90 degrees and no rain since that last snowfall. Poor man’s manure my eye, as the hay season is off to a slow start. Unpredictable is the buzz word for 2020. I guess. Who could have predicted COVID-19?. But we as deer farmers and ranchers have been thru this before. Remember 2002 and the massive shutdown of our industry due to the discovery of CWD in Wisconsin? We got thru that and in the process created a stronger and more vital industry for all. Here at NADeFA we hunkered down. We had to cancel our annual conference which generates 70% of our annual revenue. Shawn and the board contacted everyone who had prepaid for the conference and many of you donated that money to the general fund. It is very much appreciated. In addition, we applied for a PPP loan to help with salary, rent, and utility expenses. We received that money in the first round. A great big thank you to Marci for her diligence with Key Bank! As of this writing, the forgiveness applications have come out and we hope to be able to get most, if not all of the loan forgiven. We still have a long haul ahead of us, as we will still need funds to get us to next year’s conference. From the graphs, you can see how important membership has become for sustaining the organization. We are conducting a membership drive and would appreciate your help. If you know of a deer farmer who is not a member of NADeFA, encourage them to join. And don’t forget to include a membership with the deer you sell, if the buyer is not a member. Look soon for an on line auction that will replace the one that we would have had at the conference. And finally, we’re going to be at French lick for 2021 and 2022, so plan ahead and set aside the dates. And if you can, pay for the 2021 conference early. Every little bit helps. u
SECOND QUARTER 2020 INCOME
SECOND QUARTER 2020 EXPENSES
Respectfully submitted, Hank Dimuzio Treasurer NADeFA 78
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» MIDGE CONTROL
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Fungus Among Us! The potential for biting midge control By Robert “JR” Ewing and Lee W. Cohnstaedt Often, we are asked if there is a “natural” or biological control agent for biting midges. These small insects can transmit epizootic hemorrhagic disease and blue tongue viruses to cervids and are the third leading cause of deer death (after cars and bullets). In this review, we summarize the potential for reducing Culicoides biting midge numbers using entomopathic fungi. Controlling pest organisms using biocontrol agents is becoming increasingly popular by consumers due to the minimal environmental impact and the desire to use natural products that specifically target insects as a biocontrol agent. However, natural products are not without their hazards, and it must be stated that this is not an endorsement of any product; rather this article is written to inform, allowing captive cervid farmers the option to choose alternative methods that best fit the integrated pest management (IPM) strategy on their farm. As most are aware, biting midges are difficult to manage and may transmit viruses resulting in diseases that can negatively influence profits due to cervid morbidity and mortality. Of major concern are viruses vectored to deer, elk and cattle by Culicoides spp such as bluetongue (BTV) and epizootic hemorrhagic disease (EHDV), which can result in tremendous loss to the farmer/rancher. Management of pest species often involves an
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» MIDGE CONTROL « CONTINUED FROM PREVIOUS PAGE
species of fungi can cause infections in insects or mites and while they are able to attack a wide range of insects/mites, individual strains of fungi can be very specific (Abdelghany, 2015). Thus, following label rates/directions and the selection of specific strains for specific pests are key to using EPF’s as a biocontrol measure. Despite their specificity using EPF’s may cause concern when focusing on beneficial insects i.e. those whose function fall in one or more of the following categories: predators, pollinators or decomposers. Most agricultural row crops (corn, sorghum, wheat, etc.) do not rely on pollinators, but can flourish when predatory insects help manage pest species. Clover as a cover crop, and in some cases alfalfa, benefit from pollinators as well as predators thus EPF’s may be a concern to deer farmers who use these on their farms to supplement diets. This concern may be alleviated when looking at the life cycle of the insects. Adults, in general, are typically the pollinators whereas the larval stage is the destructive stage (think butterfly and caterpillar). The decomposers (mainly beetles and other arthropods) are beneficial by breaking down garbage (dung) and turning it into organic material useful for the soil. Just as with the decisions targeting the life cycle in an IPM program, life cycle decisions are made with the beneficial insects as well. Al Mazra’awi et al, used, in their 2006 study, honeybees to disseminate an EPF in large cage trials on canola. The fungus used did not result in honeybee mortality but did have significant mortality effect on the targeted pest. The success of the study centered on using a specific EPF strain against a specific pest much like using a specific insecticide to target a specific pest. Numerous studies involve the successful use of EPF’s against various mosquito species, therefore use of specific fungi to control biting midges is an idea worth considering. The ease of use with a product that needs only to be spread in areas where biting midges populate is exciting, not to mention cost effective. Ansari et al., (2019) highlighted several potential candidates in their reviewof which we will highlight three: an aquatic, Lagenidium giganteum, and the terrestrial Metarhizium anisopliae and Bassiana spp.
integrated approach possibly using chemicals, sanitation or cultural changes such as placing nets around pens or enclosures. As with any IPM program, knowledge of the pest’s lifecycle is key when selecting management tools and methods. As an example, when using pesticides to target newly emerging adults the placement of the chemical requires timing the treatment with adult emergence and knowing the preferred habitat to obtain the highest pest mortality, with the least product and damage to the environment. The weather can also place severe restrictions on pesticide use: wind, rain, sunlight and moisture can affect the efficacy and duration of chemicals. High winds increase the chance of overspray thereby affecting unintended areas. Rain can wash away freshly treated areas resulting in total loss of treatment. Too much direct sun on certain chemicals can cause them to break down quickly, thus reducing or missing a window of opportunity. Treating ponds or standing water with chemicals can have negative effects as well on non-target organisms such as fish, crustaceans, or possibly beneficial insects. The solution for killing biting midges appears, at times, out of reach. However, despite the challenges, new techniques and methods are being tested and adapted to not only better understand this pest, but to better target them. The use of novel approaches which may include biocontrol elements may be the path to success. Biocontrol methods are the use of other organisms (agents) that target pest species and attempt to reduce the local pest population. Biocontrol can center on predation the introduction of spiders in a fly infested barn, herbivory the use of plants chemical defenses to repel, or parasitism – the use of specific fungi to feed on the species to be controlled. Typically, biocontrol measures involve some form of management from a human perspective. Entomopathogenic fungi (EPF) as the name implies, are fungi which have evolved to utilize insects as a primary source of nutrients and can affect a wide range of insects in varying stages of the insect life cycle. A primary benefit of EPF when compared to other biocontrol agents is that they do not have to be ingested by the insect to cause infection. Some reports of infection through siphon tips, primarily in aquatic larvae, have occurred. However, simple contact with the reproductive spores of the fungi can be enough to LAgEniDiuM gigAnTEuM generally infect most insects (Rai et al., 2014). Nearly 750 A potential agent for use in aquatic environments is the 84
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water mold, Lagenidium giganteum Couch (Oomycetes: – Lagenidiles). Exhibiting characteristics similar to fungi, L. giganteum is an aquatic Oomycete fungal pathogen of mosquito larvae and was registered in 1995 under the trade name Laginex with the US Environmental Protection Agency (May, 2006). Studies using L. giganteum conducted with biting midge larva, Culicoides molestus, in New South Wales, Australia, resulted in up to 33% mortality in larva tested (Wright and Easton, 2007). The capability of L. giganteum to parasitize and kill (albeit slowly) both biting midge and mosquito larvae strengthen its potential in IPM programs. Nevertheless, in 2011 it was voluntarily cancelled and removed from registration by the manufacturer (Federal Register, 2011) due to infections connected with dogs, cats and humans (Vilela et al, 2015) and is not recommended for use at the time of this writing. METArHiziuM AniSopLiAE The terrestrial fungus, Metarhizium anisopliae, is one of the most widely used biocontrol agents in the world. With a worldwide distribution, M. anisopliae can be found in both the soil and on insects from the tropics to the artic (Zimmermann, 2007). As a biocontrol agent, application of the conidia (asexual spores) to different substrates: mud, leaf litter, the walls of sheds and stalls is likely to infect midge adults on resting sites. Combining M. anisopliae conidia with manure can have significant effect on adult midge emergence. Researchers obtained 98% reduction in the emergence of the biting midge, Culicoides brevitaris, adults when they mixed M. anisopliae conidia with cattle manure (Nicholas and McCorkell, 2014). As an insecticide, M. anisopliae is not necessarily as fast acting as some of the commercial pesticides commonly used to control midges. According to Zimmermann, in general, the spores, once attached to the insect cuticle, penetrate and form hyphal bodies that spread throughout the host feeding upon the nutrients in the insect hemolymph effectively starving the insect to death. Once the insect dies, conidia are produced outside the body and the cycle begins again. The entire cycle from infection to death of the insect can take days or even weeks and is dependent upon weather conditions, under dry conditions the spores may form inside rather than outside of the dead insect awaiting more favorable conditions. Largely, entomopathogenic fungi survive and are more efficacious at Spring 2020
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higher relative humidity (RH) and M. anisopliae is not an exception. The higher the RH the better chance for germination thus increasing the ability to better control the midge population. Humidity is not the only factor to consider when employing M. anisopliae as a biocontrol agent, temperature also has potential effect on fungal growth. The temperature range for M. anisopliae is moderate, generally between 15 and 35°C with optimal temperatures for germination and growth being between 25 and 35°C (Zimmermann, 2007). Another environmental concern when considering the use of M. anisopliae as a biocontrol agent is the amount of natural sunlight exposure. UV-B (280-320 nm) and UV-A (320-400 nm) are the most damaging and can cause inactivation of conidia within hours (Zimmermann, 2007). Thus, well shaded, warm and moist environments should be considered as a primary location for M. anisopliae use. BEAuVEriA BASSiAnA Another terrestrial entomopathogenic fungi, Beauveria bassiana, has similar characteristics to those of M. anisopliae. Widely known and used, especially in agricultural settings, B. bassiana has shown to be effective in the management of some Lepidopteran (moths and butterflies) and Coleopteran spp. (beetles) Liu and Bauer (2007) determined the use of B. bassinana strain GHA as a topical spray on infested ash trees was a good management tool for the emerald ash borer, Agrilus planipennis. Vandenbrg et al (1998) evaluated B. bassinana for the control of the diamondback moth, Plutella xylostella, on cabbage seedlings determining that fungus treatments had significant effect on the mortality of larva with plants in growth chambers. When used as a potential management tool for Culicoides spp, Ansari et al (2019) tested three strains of B. bassiana resulting in larval mortality of 20% and 35%. A few things to remember when using EPF: (1) Use the products only according to their product labels. The label is the law. (2) EPF’s may not be species specific and may hurt benficial insects. So, use caution when applying these products. (3) Keep in mind that entomopathic fungus is only one tool for controlling midges and all tools should be explored and adjusted to fit your farm. Remember, this is not an endorsement of a specific product, rather as stated CONTINUED ON NEXT PAGE»
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» MIDGE CONTROL A swarm of biting midges.
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previously, this is an informative review of the current literature on the potential use of three species of entomopathogenic fungi. All species outlined have been used reduce populations of mosquitos or biting midges however there are pros and cons to their use. Due diligence must be conducted when attempting to incorporate any biocontrol agent into an existing IPM program. There is no one perfect solution. Constant surveillance, awareness and utilization of effective IPM measures are required for a successful outcome. Again, follow the label for instructions on use and amount to apply. WorKS CiTED Abdelghany T. M. (2015). Entomopathogenic fungi and their role in biological control. OMICS International, 1- 42. Al Mazra’awi, M. S., Shipp, J. L., Broadbent, A. B., and Kevan P. G., (2006). Dissemination of Beauveria bassiana by honeybee (Hymenoptera: Apidae) for control of tarnished plant bug (Hemiptera: Miridae) on canola. Environmental Entomology 35(6): 1569 – 1577. Ansari, M., Walker, M., & Dyson, P. (2019). Fungi as Biocontrol Agents of Culicoides Biting Midges, the Putative Vectors of Bluetongue Disease. Vector-Borne and Zoonotic Diseases. Federal Register (2011). Product cancellation order for certain pesticide registrations. Environmental Protection agency, EPA–HQ–OPP–2009–1017; FRL–8889–7. Federal Register / Vol. 76, No. 188 / Wednesday, September 28, 2011 / Notices. https://www.govinfo.gov/content/pkg/FR-2011-0928/pdf/2011-24832.pdf
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May B.A., J.S. VanderGheynst and T. Rumsey (2006). The kinetics of Lagenidium giganteum growth in liquid and solid cultures. Journal of Applied Microbiology,101 807814, doi:10.1111/j.1365-2672.2006.02967.x Nicholas, A. and McCorkell, B. (2014), Evaluation of Metarhizium anisopliae for the control of Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae), the principal vector of bluetongue virus in Australia. Journal of Vector Ecology, 39: 213-218. doi:10.1111/j.19487134.2014.12089.x Rai, D., Updhyay, V., Mehra, P., Rana, M., & Pandey, A. K. (2014). Potential of entomopathogenic fungi as biopesticides. Indian Journal of Science Research and Technology, 2(5), 7-13. Vandenberg, J. D., Shelton, A. M., Wilsey, W. T., and Ramos M (1998) Assessment of Beauveria bassiana sprays for control of diamondback moth (Lepidoptera: Plutellidae) on crucifers. Journal of Economic Entomology, 91(3): 624630 Vilela, R., Taylor, J. W., Walker, E. D., & Mendoza, L. (2015). Lagenidium giganteum pathogenicity in mammals. Emerging infectious diseases, 21(2), 290–297. doi:10.3201/eid2102.141091Wright, P. J., and S. Easton, C. (2007). Natural Incidence of Lagenidium giganteum Couch (Oomycetes: Lagenidiales) Infecting the Biting Midge Culicoides molestus (Skuse) (Diptera: Ceratopogonidae). Australian Journal of Entomology, 35. 131 - 134. 10.1111/j.1440-6055.1996.tb01376.x. Zimmermann, G. (2007) Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology, 17:9, 879-920, DOI: 10.1080/09583150701593963 u nADeFA.org
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» WILDLIFE CAPTURE
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The Evolution of
WILDLIFE ANESTHESIA By Wildlife Pharmaceuticals Chemical restraint and anesthesia of wildlife has come a long way since primitive South American tribes used curare-coated arrows or blow pipe darts to immobilize animals when hunting for food. These and other outmoded methods of capture were relied upon for centuries. During the early 1950’s wildlife biologists and veterinarians began exploring the use of newly-developed neuromuscular blocking drugs that enabled immobilization through the control of muscles at the neuromuscular junction. From this initial success of formalized chemical immobilization procedures, field capture methods of wildlife species have evolved rapidly over decades. While immobilization of deer with these neuromuscular blocking drugs, like tubocurarine, gallamine, and succinylcholine had been reported as early as 1953, extensive negative side effects and high deer mortality limited their use. The search for better immobilization drugs options then focused on finding a solution that would be: • safe, effective and efficient • easy to administer in small dose volumes • have a rapid onset with predictable action • be quickly reversible • and provide rapid and uneventful recovery By the 1970’s wildlife veterinarians began exploring the use of ultrapotent opioids, such as etorphine and later carfentanil and thiafentanil. Although very effective in their use, the human risks of handling these drugs convinced wildlife professionals to pursue alternative immobilization agents. Turning to a new class of drugs, the alpha 2 agonists that produce predictable, dose-dependent sedation and analgesia. These formulations could be administered via remote darting without causing irritation, and most importantly, responded to specific reversal agents that enabled rapid recovery.
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Since the α-2 Adrenergic drug receptors are located with opioid drug receptors, they also found that by combining agents from these two drug classes resulted in a highly desirable synergistic form of sedation and analgesia. Discovery of such alpha-2 agonist/benzodiazepine mixtures as tiletamine/zolazepam and xylazine hydrochloride administered intramuscularly, provided reasonable onset of anesthesia in both captive and free-ranging cervids. This combination quickly became the most common drug protocol used to immobilize deer. However, while considered one of the most suitable combinations over the next 30 years, this tiletamine/zolazepam and xylazine drug protocol still had some major disadvantages. While effectively immobilizing many species of deer it also resulted in such life-threatening side effects as bloating, regurgitation, rumen disorders, lowered oxygen levels, rising body temperatures, heart irregularities and breathing problems. DesireD CharaCteristiCs of effeCtive WilDlife anesthesia Since the performance objectives for developing a new anesthetic combination required improved efficacy and safety qualities over existing drug protocols, researchers needed to create an immobilization formulation that provided: • effective tranquilization (drug induced, calm state without drowsiness) • rapid sedation (fast onset of action) • anxiolyxsis (loss of fear and “anxiety”) • excellent analgesia (freedom from pain) • efficient reversibility (on demand) • no lingering side effects CONTINUED ON NEXT PAGE»
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» WILDLIFE CAPTURE
Table 1.
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BuTorpHAnoL • An opiate agonist/antagonist with a DEA Class IV Given some of the disadvantages reported with previous restriction anesthetic combinations and consideration of the above • A totally synthetic, centrally acting, narcotic agonistcharacteristics, a search for better alternatives continued. While investigating new anesthesia options, Dr. Lisa antagonist analgesic Wolfe and Dr. William Lance explored the use of an unprecedented drug combination containing butorphanol, AzApEronE • A short-acting neuroleptic sedative previously used azaperone and medetomidine. Initial mule deer trials were to reduce stress from capture and handling in a number of conducted and reported highly positive results. With expanded testing in other deer species, this patented species • Most commonly used previously in Africa with formulation (abbreviated as the acronym BAM), continued to demonstrate exceptional effectiveness for immobilization Etorphine (M99) and Thiafentanil (A3080) or alone as an of deer based on its unique synergistic central nervous aid to help transport hoof stock system mode of action. Ongoing field research among many different wildlife MEDEToMiDinE • Although previously used in wildlife with and exotic species continued to confirm how this innovative combination of select pharmaceuticals, brings out the best Carfentanil, A3080, Ketamine and Telazol its combination attributes of each drug shown below, at the lowest effective with Butorphanol and Azaperone was a novel concept. • Selecting this site-specific alpha-2 agonist dose rate. Here’s a summary overview of the components that medetomidine proved to provide a safer, more potent alternative to xylazine make up this reversible field anesthesia, BAM.
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• In addition, it could be quickly and completely reversed by the alpha-2 antagonist atipamezole. raiSing THE STAnDArD oF AnESTHESiA For WiLDLiFE iMMoBiLizATion Since prior to the development of BAM, use of the tiletamine/zolazepam and xylazine drug protocol was the choice of most wildlife veterinarians and captive deer farmers, it was important to find out how this new butorphanol-azaperone-medetomidine combination compared. Shown on left page in Table 1, are the key benchmarks for measuring an effective immobilization process. While similarities were reported in the induction times of both formulations, note the major differences in such critical factors as respiratory response, body temperature, reversal requirements, dosage administration and safety margins. BAM use then continued to increase beyond white tail deer immobilizations, expanding rapidly to include effective use in fallow deer, axis deer, elk, caribou, reindeer, moose, bison and other hoof stock species. Over the last decade this reversible anesthesia combination has now been successfully used in over 60 species. With the continued growth in exotic game ranching, BAM has quickly become the anesthesia of choice for immobilizing such sensitive species as kudu, sable, gemsbok, wildebeest, blackbuck, Arabian oryx, Scimitar horned oryx
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and impala. Literally thousands of exotics and non-domestic animals have been safely anesthetized using BAM with excellent results. uSE oF BAM For LApAroSCopiC ArTiFiCiAL inSEMinATion in DEEr While BAM use for immobilization was steadily advancing among deer farmers for conducting most routine procedures (vaccinations, cutting antlers, giving antibiotics, etc.), its administration as an effective anesthesia for use during laparoscopic artificial insemination (Lap AI) was unknown. Use of tiletamine HCl /Zolazepam HCl - Xylazine (Tel/Xyl) continued as the preferred drug combination to provide the anesthetic induction level required for LAP AI. In order to determine if BAM would be a suitable alternative to anesthetize does undergoing LAP AI, a formal study was conducted in 2013 to compare the anesthesia effectiveness of BAM versus the Telazol-xylazine combination. The study was done by randomly assigning forty-two white tailed does to one of two treatment groups. Group 1 received the Tel/Xyl injection protocol, while Group 2 animals were injected with BAM. Data was collected to measure the effects these two different protocols had on quality of anesthesia and conception rates following CONTINUED ON NEXT PAGE»
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» WILDLIFE CAPTURE
Figure 1. Based on an established Reversal Condition rating system*, overall quality of recovery from BAM anesthesia was judged to be superior at all recorded time intervals, (observed at 5, 10, 15, 30, 45, 60 and 90 minutes post-reversal).
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To evaluate the quality of anesthesia produced by a laparoscopic artificial insemination. In addition, combination of butorphanol-azaperone-medetomidine measurements for time to induction (first approach); time (BAM), as compared to Telazol / Xylazine a comparative to reversal; as well as overall quality of recovery were study was conducted in 2017. This study was designed to determine if BAM could provide the plane of anesthesia recorded and compared. required for successful semen collection from white-tailed bucks, as compared to using the traditional Telazol / rESuLTS oF THiS STuDY, rEporTED THE Xylazine combination. Beyond the ability to collect semen FoLLoWing: • AI conception rates of ~55% reported among does in from each buck, it was equally important to identify any variations in quality of semen collected when using these two both BAM and Tel/Xyl treatment groups different anesthetic protocols. o BAM Group Does: 12 Pregnant and 10 Open o T/X Group Does: 11 Pregnant and 9 Open A group of ten bucks were selected for the study, based • The BAM treatment group demonstrated a on their history of successfully breeding between 10-30 does significantly improved quality of recovery and a greatly per season on a regular basis. All animals were anesthetized reduced duration of sedation once in January with Telazol / Xylazine (Tel/Xyl) then again o 46% of BAM treated does presented no sign of following a 4-week washout period, in February using sedation within 15 minutes of reversal (Figure 1) butorphanol-azaperone-medetomidine (BAM). While under anesthesia semen was collected each time using a uSE oF BAM For SuCCESSFuL SEMEn standard deer electroejaculation collection procedure. CoLLECTion in WHiTE TAiLED BuCKS Following each semen collection phase, all ejaculate Although the 2013 Lap AI study provided veterinarians samples were analyzed for sperm quality using a and wildlife reproduction professionals with the evidence Computerized Assisted Sperm Analysis (CASA) system. needed for confidence to use BAM for insemination of does, Results were recorded measuring: total semen volume, many questions were raised as to its suitability for the sperm concentration and total sperm numbers, as well as successful collection of semen from white tailed bucks. sperm motility factors. Again, the Telazol / Xylazine combination had long been the anesthetic standard preferred by most deer farms for CoMpAraTiVE STuDY rESuLTS rEporTED: extracting semen. • BAM anesthesia provided equal or increased semen 94
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Table 2
collection volumes as compared to Telazol / Xylazine. SuMMArY • Semen from test group males immobilized with As the science of anesthesia has evolved from its BAM displayed all important traits needed for successful primitive origins in the jungles of South America, numerous fertilization: level of sperm concentration; total sperm new capture methods and drug protocols have been number; and sperm motility. developed and used for different wildlife species. Improvement of chemical immobilization formulations has When evaluating the quality of anesthesia induction and recovery, it was shown that while under BAM anesthesia the played a major role in improving animal welfare, minimizing average breaths per minute of bucks was 21% lower than animal distress through lowering the incidence of discomfort while they were under Telazol / Xylazine. Following and death. Since the development of the unrivaled butorphanolcollection the comparison of reversal times recorded averaged just 3.5 minutes using BAM as compared to 48.3 azaperone-medetomidine (BAM) combination, wildlife minutes when reversing Telazol / Xylazine. Table 2 above veterinarians and deer management professionals now shows a summary average of the differences in vital sign depend on this fully-reversible anesthesia for rapid, smooth indicators and recovery times when comparing BAM to inductions; relaxed respiration; little increase in body Tel/Xyl anesthesia. temperature; healthy blood oxygen levels; comforting The results of this 2017 study suggest that use of BAM immobilization of bucks provides effective anesthesia for relaxation; excellent pain control and smooth recoveries. equivalent and somewhat improved semen collection in Plus, it has been proven effective for both laparoscopic white-tailed bucks as compared to the traditional Telazol / artificial insemination and semen collection through Xylazine combination. electroejaculation. u
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Âť EHD RESEARCH
Medgene Labsâ&#x20AC;&#x2122; EHDV Vaccine Update By Ashley Petersen, Clinical Research & Development Lead Scientist, Medgene Labs In 2019, Medgene Labs was allowed to evaluate an experimental EHDV2 vaccine across the United States. Deer farms in Florida, Illinois, Louisiana, Kansas, Iowa, South Dakota, North Dakota, and Oklahoma participated in the vaccine evaluation. In Florida, Medgene worked with the Cervidae Health and Research Initiative (CHeRI) to monitor the use of the vaccine and conduct diagnostics on deer that died on farms participating in the trial. Fourteen farms in Florida received the vaccine. Across those farms, CHeRI necropsied 34 deer and determined the cause of death. Table 1 includes details
regarding the cause of death and whether or not the deer was vaccinated with the experimental EHDV2 vaccine. It was determined that no vaccinated deer died from EHDV2 infection. See Table 1. Florida Deer necropsy Summary In other states, Medgene collected data on the safety of the vaccine. Users were asked to record all adverse reactions (AEs) and report them to Medgene at the end of 2019. Table 2 shows the observed AEs and the number of farms that reported each specific AE. Florida farms were also included in the summary. All AEs were reported to resolve within a
Table 1. Florida Deer necropsy Summary VACCinATED (Y/n)? n n n n n n n n n n n n n n n n n Y n n n n Y Y n Y Y Y Y Y n Y Y Y
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CAuSE oF DEATH EHDV2 infection and secondary bacterial septicemia. Bacterial pneumonia. EHDV2 infection. Bacterial pneumonia. Bacterial pneumonia. Antler infection. EHDV infection and secondary bacterial septicemia. Bacterial sepsis. Physical trauma. EHDV2 infection. Bacterial pneumonia. Bacterial pneumonia. EHDV infection and bacterial pneumonia. EHDV2 infection. Unknown. Co-infection of EHDV and BTV. Bacterial pneumonia. BTV infection. Bacterial and viral pneumonia. Bacterial infection. EHDV1 infection. Bacterial pneumonia. EHDV2 infection. Deer received vaccine one day prior to death. EHDV2 infection was already present at time of vaccination. Bacterial infection. Bacterial sepsis. Unknown. Tested negative for EHDV and BTV. EHDV6 infection and bacterial pneumonia. EHDV1 infection. EHDV1 infection and bacterial sepsis. EHDV1 infection. EHDV1 infection. EHDV1 infection and bronchopneumonia. Unknown. Tested negative for EHDV and BTV. Unknown. Tested negative for EHDV and BTV.
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IMAGE HERE
George Courtney • 682-229-7008 • george.courtney12@gmail.com Spring 2020
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» EHD RESEARCH Table 2. Adverse reaction Summary rEporTED ADVErSE rEACTion
nuMBEr oF FArMS
None
11
Lameness/limping
4
Lethargy
2
Fever
2
Injection Site Reaction
2
Off feed
1
Table 3. EHDV2 Antibody Titers in Schafer Herd Deer
pre-vaccination, nov2019
1 Dose
2 Doses
Table 4. EHDV2 Antibody Titers in Sheep Vaccine (EHDV Serotype)
Animal iD
Titer ≥320
9027
NA P901
10
≥320
≥320 9031
NA P918
≥320
80
160
40
≥320
9083
10
≥320
9093
40
NA
40
NA
1, 2, & 6 bottled together
9034
NA Gr14
≥320
NA
80
Gr23
≥320
NA Or85
83 ≥320
NA
750
Or91
≥320 one dose of each (3 separate shots)
NA B33
<10
3416
NA
≥320 9032
NA B34
10
NA
40 9066
NA Y28
<10
≥320
20
NA
20
NA
<10
NA
NA
≥320
≥320 5
NA P4 NA G21
≥320 65 1, 2, & 6 bottled separately, combined into one before vaccination
≥320
NA G6
9061 ≥320
<10 R828*
20 9038
9080 ≥320 9026
NA R819*
NA
≥320
80 9040
NA R811*
NA
≥320
160 9049
2 alone
10 R832*
NA
≥320
NA
≥320
≥320 9050
20 R835*
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≥320 9075
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graph 1. Summary of Sheep Titers to EHDV2
week of vaccine administration, with deer returning to normal health. The observed reactions are all typical events that occur after working and vaccinating large animals. No farms that properly used the experimental EHDV2 vaccine reported a deer death attributed to EHDV2 infection. See Table 2. Adverse reaction Summary The study data presented above has been shared with the Center for Veterinary Biologics (CVB) at the United States Department of Agriculture (USDA). CVB is the governing agency that licenses veterinary biologics and to whom Medgene reports all study data and vaccine usage. Further vaccine evaluation in 2019 included serology testing. Executive director of NADeFA, Shawn Schafer, worked with Medgene to monitor EHDV2 antibody titers in some of his vaccinated deer. Table 3 summarizes the findings. Shawn drew blood samples on randomly selected deer at three diďŹ&#x20AC;erent times. Deer marked with an asterisk were previously vaccinated with Medgeneâ&#x20AC;&#x2122;s EHDV2 vaccine in December 2018. In can be concluded that after two doses of the experimental EHDV2 vaccine, deer elicited an immune response to the vaccine.
performed for EHDV1 and EHDV6 antibody tests. Therefore, Table 4 and Graph 1 depict antibody titers only to EHDV2. Each animal received two doses of vaccine, given 3 weeks apart. The titers reported in Table 4 and Graph 1 were from two weeks post second vaccination. Results indicated that in all test groups with EHDV2 in the vaccine, deer generated antibodies to EHDV2. See Table 4. EHDV2 Antibody Titers in Sheep See graph 1. Summary of Sheep Titers to EHDV2
The apparent success of the experimental EHDV2 vaccine and the promising results of a multivalent EHD vaccine led Medgene to request, and ultimately receive, permission to conduct a field evaluation of an experimental multivalent EHDV2 & 6 vaccine in 2020. Because EHDV2 and EHDV6 are the most prevalent Serotypes in the U.S., and due to wanting to step-wise add each Serotype to one vaccine, EHDV1 was not included for evaluation in 2020. The vaccine evaluation includes use in white-tailed deer, bison, elk, fallow deer, goats, moose, mule deer, muntjac deer, red deer, reindeer, sheep, and sika deer. The experimental vaccine is available directly from Medgene See Table 3. EHDV2 Antibody Titers in Schafer Herd Labs. For more information about evaluating the vaccine in herd, please contact Ashley at In tandem with the vaccine trials in white-tailed deer, your Medgene Labs conducted a study in sheep. This study was ashley@medgenelabs.com or 605-692-1268. Medgene Labs would like to thank CHeRI, NADeFA, designed to evaluate the use of vaccines for EHDV1, EHDV2, and EHDV6 in combination and apart. Medgene and all producers that evaluated the EHDV2 vaccine in 2019. Labs has the ability to accurately and consistently test We look forward to continued collaboration in 2020 and aim antibody titers to EHDV2. Currently, optimization is being to provide further success in combating EHDV. u Spring 2020
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Accurate Genomic Predictions for Chronic Wasting Disease in U.S. White-Tailed Deer Christopher M. Seabury,*,1 David L. Oldeschulte,* Eric K. Bhattarai,* Dhruti Legare,† Pamela J. Ferro,† Richard P. Metz,‡ Charles D. Johnson,‡ Mitchell A. Lockwood,§ and Tracy A. Nichols** *Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, †Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, Texas, ‡Genomics Core, Texas A&M AgriLife Research, College Station, Texas, §Texas Parks and Wildlife Department, Austin, Texas, and **USDA-APHIS-VS-Cervid Health Program, Fort Collins, CO
ABSTRACT The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable (h2 ¼ 0:337 6 0:079 ─ 0:637 6 0:070Þ among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a largeeffect risk locus (P-value , 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE $ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP. Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. whitetailed deer produced mean genomic prediction accuracies $ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.
The fatal wasting syndrome known as chronic wasting disease (CWD) was first observed among captive mule deer (Odocoileus hemionus) and black-tailed deer (Odocoileus hemionus columbianus) within several Colorado wildlife facilities during the late 1960s, and histologically recognized as a prion disease by the late 1970s (Williams and Young 1980; Moreno and Telling 2018). Thereafter, CWD was described in free-ranging U.S. mule deer, elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus; hereafter WTD) and moose (Alces alces shirasi), with subsequent diagnostic surveillance suggesting an irreversible geographic expansion of the disease among farmed and free-ranging populations of these species (Moreno and Telling 2018; Gavin et al. 2019; Osterholm et al. 2019). Copyright © 2020 Seabury et al. doi: https://doi.org/10.1534/g3.119.401002 Manuscript received December 17, 2019; accepted for publication February 25, 2020; published Early Online March 2, 2020. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supplemental material available at dryad: https://doi.org/10.5061/dryad.xd2547dcw. 1 Corresponding author: E-mail: cseabury@cvm.tamu.edu
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KEYWORDS
genome-wide association chronic wasting disease white-tailed deer genomic prediction heritability
At present, at least 26 U.S. states and multiple Canadian provinces are known to be affected by CWD (Moreno and Telling 2018; Gavin et al. 2019; Osterholm et al. 2019). Likewise, Norway, Finland, and the Republic of Korea have also reported CWD in free-ranging reindeer (Rangifer tarandus; Norway), moose (Norway, Finland), and imported elk (Korea) (Moreno and Telling 2018; Gavin et al. 2019; Osterholm et al. 2019). The implementation of modern best management practices, including containment and depopulation of positive herds, has not prevented the emergence of CWD in new geographic areas (Moreno and Telling 2018; Gavin et al. 2019; Osterholm et al. 2019). Therefore, a need currently exists to develop novel strategies to reduce the prevalence of CWD among farmed deer and elk. MATERIALS AND METHODS Study overview Herein, we investigate differential susceptibility to CWD among farmed U.S. WTD by utilizing genomic DNA samples from CWD positive and CWD non-detect WTD to perform next-generation sequencing with variant prediction for the construction and validation of a medium density SNP array. Thereafter, we use the array in conjunction with PRNP genotypes to conduct genome-wide association
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analyses (GWAA’s) and produce marker-based heritability estimates. Finally, we conclude our study by utilizing the genome-wide SNP data to deploy genomic prediction equations with k-fold cross validation to assess the potential for developing a genomically-estimated CWD eradication program. Animal resources, CWD diagnostics, and DNA isolation Frozen whole blood samples (n = 448) and rectal biopsies (n = 37) from farmed U.S. white-tailed deer (WTD; both sexes) were available within an existing USDA APHIS repository that was created via federal CWD surveillance activities; including depopulations of CWD positive herds (USDA APHIS, Fort Collins, CO). All herds included both CWD positive (n = 256) and CWD non-detect (n = 229) WTD (Thomsen et al. 2012), with geographic representation that included WTD farms located in the U.S. Midwest, Northeast, and South. All diagnostic classifications were based upon immunohistochemistry (i.e., IHC of lymph node, obex), as implemented and performed at USDA National Veterinary Services Laboratory (NVSL) in Ames Iowa (Thomsen et al. 2012). Genomic DNA was isolated from frozen whole blood using the Applied Biosystems MagMAX DNA MultiSample Ultra Kit with the KingFisher 96 Purification System (ThermoFisher), as recommended by the manufacturer, at the Texas Veterinary Medical Diagnostic Laboratory (TVMDL; College Station, TX). Genomic DNA was isolated from WTD rectal biopsies using the LGC sbeadex tissue purification kit (LGC) with automation at GeneSeek Neogen (Lincoln, NE). Hair samples (n . 700) from farmed WTD (both sexes) were also available within an existing Texas Parks and Wildlife Department (TPWD; Austin, TX) repository created via surveillance and depopulation efforts after the initial detection of CWD in Texas. At the time of study, sample repositories for these herds included CWD positive (n $ 100) and CWD nondetect (n $ 600) WTD, with a geographic representation that included WTD farms located in the U.S. South (Texas). All diagnostic classifications for TPWD samples were based upon IHC (i.e., lymph node, obex, and one tonsil) initially performed at TVMDL, with further confirmation at NVSL (Thomsen et al. 2012). Genomic DNA was isolated from WTD hair follicles using the LGC sbeadex tissue purification kit (LGC) with automation at GeneSeek Neogen (Lincoln, NE). All genomic DNAs were quantified and evaluated for purity (260/280 ratio) via Nanodrop (ThermoFisher). Reduced representation libraries and sequencing Pooled DNA samples were previously shown to be effective for variant prediction; thus enabling downstream genotyping in WTD (Seabury et al. 2011). Therefore, pooled DNA samples were created for CWD positive (WT1) and CWD non-detect (WT0) WTD acquired from the USDA APHIS repository (frozen blood). Briefly, WT1 (n = 190) and WT0 (n = 184) genomic DNAs with concentrations $ 15 ng / ml were used to construct sequencing pools representing depopulated WTD from the U.S. Midwest, Northeast, and South by targeting 50 ng of genomic DNA per WTD in each respective pool (WT1, WT0). Genomic DNAs with concentrations , 15 ng / ml were retained for downstream Affymetrix Axiom and PRNP genotyping. Aliquots from each genomic DNA pool (WT0, WT1) were digested with EcoRI, HindIII, and PstI (NEB) in 1X CutSmart buffer for 4 hr at 37 . Enzymes were heat inactivated at 80 for 20 min and held at 10 until ligation. Ligase buffer, ligase (NEB) and barcoded enzyme-specific adapters compatible with DNA possessing EcoRI, HindIII or PstI overhangs were added to the digested samples, and incubated at 16 for 8 hr. Following heat inactivation at 80 for 20 min, 1/10th volume
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of 3M NaAc (pH 5.2) and two volumes of 100% ethanol were added to each sample, and then held at -20 for 1 hr before spinning at high speed for 10 min in a bench-top microfuge. Pellets were washed twice in 1 ml 70% ethanol and resuspended in 130 ml 1X TE. Samples were then sheared to an average size of 350 bp on the Covaris E220 sonicator, and AMPure XP bead purified as per the manufacturers protocol (Beckman Coulter). Sheared DNA fragments were size selected using a Pippin Prep 2% dye-free agarose gel with internal size markers (Sage Bioscience); aiming for 300-800 bp inserts. Recovered samples were cleaned with 1X AMPure XP beads and endrepaired first with Bst DNA Polymerase (NEB), then with a DNA End Repair Kit (NEB), and A-Tailed using Klenow Fragment (39/59 exo-) (NEB) in the presence of 50 nM ATP. An Illumina P7-adapter (Adapter B) was ligated to the A-tailed ends as described above. Following two rounds of AMPure XP bead purification, 150 ng of each pool was then subjected to “pre-selection PCR” (PreCR) in which a biotinylated forward primer (P5-Select) and unique indexed reverse primers (TDX) were used to amplify and tag desired DNA fragments. Reactions (200ml total) contained 200 nM dNTPs, biotinylated forward and two P7-index primers per pool, 4 units Q5 Hi-Fidelity Taq (NEB), and were split into 2 X 100 ml volumes for thermocycling. Following an initial denaturation at 98 for 30 sec, samples were subjected to 15 cycles of 98 for 10 sec, 72 for 30 sec then a final elongation at 72 for 5 min and held at 4 . PCR products were purified using Qiagen PCR purification columns, then 1X AMPure XP beads, and quantified via DeNovix. Removal of undesirable fragments (P5 to P5 and P7 to P7 ligated products) was achieved with Dynabeads M-270 Streptavidin coupled magnetic beads (ThermoFisher). Briefly, 50 ml of beads per sample were captured and washed twice with 1X Bead Washing Buffer (1X BWB, 10 mM Tris-HCl with pH 7.5, 1 mM EDTA, 2 M NaCl). Beads were resuspended in 100ml 2X BWB and mixed with 2000 ng of PreCR product in 100 ml EB. After 20 min at room temperature, beads were captured and washed three times in 200 ml 1X BWB, twice in 200 ml water, and once in 100 ml 1X SSC. Beads were then resuspended in 50 ml 1X SSC, heated at 98 for 5 min, and placed on a magnet, with the supernatant removed thereafter. This elution was repeated and the final supernatants were purified with Qiagen PCR columns, as recommended by the manufacturer. The eluted ssDNA was DeNovix quantified, and diluted to 1 ng/ml with EB. A final PCR was performed on 10 ng of input DNA using FiLi-F1 and FiLi-R1 primers in a 50 ml reaction, with only 8 cycles. Final PCR products representing WT0-EcoRI, WT0-HindIII, WT0-PstI, WT1EcoRI, WT1-HindIII, and WT1-PstI were purified with 1X AMPure XP beads, quantified and assessed for quality on a Fragment Analyzer (Advanced Analytics), and sequenced (2 · 125 bp, paired end) on the Illumina HiSeq 2500 at the Texas AgriLife Genomics and Bioinformatics Core Facility at Texas A&M University. Raw reads generated for each library were as follows: WT1 EcoRI (134,299,714); WT1 PstI (175,412,740); WT1 HindIII (152,371,052); WT0 EcoRI (145,989,752); WT0 PstI (120,598,450); WT0 HindIII (137,148,058). Primers used were as follows: For ligation to restriction enzyme cut DNA, adapters were made by mixing equimolar amounts of top (T) and bottom (B) oligos in 1X oligo hybridization buffer (50 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), heating them to 98 for 1 min, and allowing them to cool to room temperature at a rate of 0.1 per second. Primer sequences used were as follows (X denotes bases used for barcoding): Eco_T,59-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACX XXX XXX XAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-39; Eco_B,59-AAT TAG ATC GGA AGA GCG TCG TGT AGG GAA AGA GTG TXX XXX XXX GTG
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TAG ATC TCG GTG GTC GCC GTA TCA TT-39; Hind_T, 59-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACX XXX XXX XAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-39; Hind_B, 59-AGC TAG ATC GGA AGA GCG TCG TGT AGG GAA AGA GTG TXX XXX XXX GTG TAG ATC TCG GTG GTC GCC GTA TCA TT-39; Pst_T, 59-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACX XXX XXX XAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC TTG CA-39; Pst_B, 59- AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT XXX XXX XXG TGT AGA TCT CGG TGG TCG CCG TAT CAT T-39; Adaptor-B_T, /5Phos/GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-39; AdaptorB_B, 59-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-39; P5_Select, /5BiotinTEG/AAT GAT ACG GCG ACC ACC GAG ATC TAC AC-39; FiLi-F1: 59-AAT GAT ACG GCG ACC ACC GAG ATC TAC AC-39; FILi-R1: 59-CAA GCA GAA GAC GGC ATA CGA GAT-39; TDX, 59-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX XGT GAC TGG AGT TCA-39. Sequence analysis and affymetrix axiom array design All Illumina sequences were trimmed for quality and adapters using CLC Genomics Workbench 10.1.1 (Qiagen), as previously described (Halley et al. 2014; Halley et al. 2015; Sollars et al. 2017). All trimmed reads were mapped to the WTD genome assembly (GCF_002102435.1 Ovir.te_1.0; https://www.ncbi.nlm.nih.gov/assembly/GCF_002102435.1/) using the CLC Genomics Workbench 10.1.1 reference mapping algorithm (Seabury et al. 2011; Halley et al. 2014; Halley et al. 2015; Sollars et al. 2017). Variant prediction was performed using a probabilistic approach implemented within CLC Genomics Workbench 10.1.1 (Halley et al. 2014; Halley et al. 2015; Oldeschulte et al. 2017; Sollars et al. 2017). This algorithm estimates error probabilities from quality scores, and uses these probabilities to determine the most likely allele combination per nucleotide position, thus facilitating a user-specified minimum probability threshold (P $ 0.95) for variant prediction, and variant quality scores (Halley et al. 2014; Halley et al. 2015; Oldeschulte et al. 2017; Sollars et al. 2017). Additional variant prediction parameters and filters were similar to those recently described (Seabury et al. 2011; Oldeschulte et al. 2017), and the probabilistic approach produced evidence for 6,268,706 variants, which included 5,561,550 putative SNPs (P $ 0.95; Minor Allele Frequency $ 0.01). Variant prediction results were exported from CLC as a single variant call formatted file (VCF), which was used for SNP array design. Briefly, the VCF file was filtered according to the Affymetrix Axiom myDesign guidelines for SNP submission (http:// www.affymetrix.com/support/technical/byproduct.affx?product= axiom_custom_agrigenomics) using a custom python script as follows: Retain only biallelic SNPs with minimum depth = 10, maximum depth = 150, minimum minor allele frequency (MAF) $ 0.15, minimum SNP quality score = 30, identify probe overlaps for exclusion, and prioritize variants that maximize array density (i.e., A/T and C/G take up two spaces on the array). The python script as well as more detailed documentation are available in Additional File 30 (DRYAD). The targeted number of SNPs for submission to Affymetrix was . 200,000; to facilitate internal Affymetrix scoring (i.e., by pconvert, best_pconvert; recommended, neutral, or not recommended) that would enable the design of a final Affymetrix Axiom 200K SNP array. Collectively, 200,000 SNPs were favorably scored (n = 179,508 recommended; n = 20, 492 neutral) and used for array fabrication. PRNP and affymetrix axiom array genotyping PRNP genotyping for missense variants at codons 37, 95, 96, 116, and 226 was performed at GeneSeek Neogen (Lincoln, NE) as part of an
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existing commercial genotyping by sequencing (GBS) service. Briefly, the functional PRNP gene was PCR amplified using primers designed to be exclusionary to a processed pseudogene as previously described (O’Rourke et al. 2004), and the resulting amplicons were purified via AMPure XP beads as recommended by the manufacturer (Beckman Coulter); thus allowing for the creation of barcoded Illumina Nextera XT DNA libraries and amplicon sequencing on an Illumina MiSeq. PRNP genotypes were called from the aligned read pileups at GeneSeek Neogen, and delivered in text format. Affymetrix Axiom 200K genotyping was also performed at GeneSeek Neogen using the established Affymetrix best practices workflow; with genotypes delivered in text format. Affymetrix quality control thresholds were DQC $ 0.82, QC call rate $ 97%, passing samples in the project $ 95%, and average call rate for passing samples $ 97%. Collectively, 860 WTD samples with the desired metadata (i.e., sex, age, U.S. general region) passed all Affymetrix QC filters; each with 125,968 SNP array genotypes, and paired PRNP genotypes, thus yielding a combined set of 125,973 SNP genotypes for analysis. Fifty-three WTD did not have CWD diagnostic data at the time of study. SNPs which did not convert on the Affymetrix Axiom 200K WTD array were primarily due to call rates below the best practices threshold (n = 37,197), and failures to meet thresholds in two or more best practices criteria (n = 36,045). All SNP conversion types are comprehensively summarized in DRYAD (Additional File 32). GWAA and genomic prediction with cross validation Prior to all analyses, a comparative marker map was created by aligning the WTD PRNP sequence and all Affymetrix Axiom 200K probe sequences with ARS-UCD1.2 (GCA_002263795.2) via blastn, thus providing comparative evidence for the origin of the array SNPs (i.e., autosomal vs. non-autosomal); which was necessary because the draft de novo WTD genome assembly (GCF_ 002102435.1 Ovir.te_1.0) is unanchored (i.e., by maps or in situ hybridization). The comparative marker map was joined to the combined set of all genotypes (PRNP + Affymetrix Axiom array), and quality control analyses were performed in SVS v8.8.2 or v8.8.3 (Golden Helix). Initially, pairwise IBS distances were computed to identify twins and duplicate samples. Eight samples present in both repositories (USDA APHIS, TPWD) were purposely duplicated for use as process controls, and correctly identified by IBS/IBD estimates (Additional File 31 in DRYAD). Eight additional samples were also identified as either duplicates or potential twins. In all cases, only the sample with the highest call rate was retained for further analyses. Additional quality control analyses and filtering were as follows: sample call rate (, 97% excluded), and thereafter, SNP filtering by call rate (. 15% missing excluded), MAF (, 0.01 excluded), polymorphism (monomorphic SNPs excluded), and Hardy-Weinberg Equilibrium (excludes SNPs with HWE P-value , 1e-25), which yielded 123,987 SNPs for all analyses. PRNP SNPs which failed to endure quality control filtering included codons 95 (MAF , 0.01) and 116 (monomorphic), whereas codons 37, 96, and 226 remained. All GWAA’s and genomic predictions with k-fold cross validation were performed on 807 WTD with recorded metadata that included sex, age, U.S. general region of origin (i.e., Midwest, Northeast, South), and CWD diagnostics. CWD phenotypes used in all analyses were: CWD Scores (0 = non-detect, 1 = lymph node positive, 2 = lymph node and obex positive); CWD Binary (0 = non-detect, 1 = lymph node positive and/or obex positive). However, at the time of study, one WTD (sample ID: MQ6Q) only possessed diagnostic data for a
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CWD positive tonsil biopsy, and thus was assigned a CWD Score and a CWD Binary phenotype of “1”. All WTD GWAA’s were performed using a mixed linear model with variance component estimates, as described and implemented in EMMAX, and executed in SVS v8.8.2 or v8.8.3 (Golden Helix), where all genotypes are also recoded as 0, 1, or 2, based on the incidence of the minor allele (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). Briefly, the general mixed model can be specified as: y ¼ Xb þ Zu þ e, where y represents a n · 1 vector of observed CWD phenotypes, X is a n · f matrix of fixed effects, b is a f · 1 vector representing the coefficients of the fixed effects, u represents the unknown random effect, and Z is a n · t matrix relating the random effect to the CWD phenotypes of interest (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). Herein, it is necessary to assume that VarðuÞ ¼ s2g K and VarðeÞ ¼ s2e I, whereby VarðyÞ ¼ s2g ZKZ ’ þ s2e I, but in this study Z represents the identity matrix I, and K represents a relationship matrix of all WTD samples. To solve the mixed model equation using a generalized least squares approach, we must first estimate the relevant variance components (i.e., s2g and s2e ) as previously described (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). Variance components were estimated using the REML-based (restricted maximum likelihood) EMMA approach (Kang et al. 2010), with stratification accounted for and controlled using a genomic relationship matrix ðG) (VanRaden 2008), as computed from the WTD genotypes. Genomic relationship matrix (GRM) heritability estimates (½h2 ¼ s2g = ðs2g þ s2e Þ ) were produced as previously described (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). Moreover, because previous WTD studies indicate that the probability of CWD infection increases with age (Grear et al. 2006), and that CWD may disparately affect male and female WTD in different U.S regions, including differences in clinical disease progression and mortality (Grear et al. 2006; Edmunds et al. 2016), the possibility for different CWD strains must be considered (Bistaffa et al. 2019). Therefore, the following fixed-effect covariates were specified for comparison of GWAAs: sex, age, U.S. region of origin, and the total number as well as types (0 = none-detected; 1 = lymph node only; 2 = lymph node and obex) of CWD positive diagnostic tissues, with one exception (i.e., sample ID: MQ6Q), as noted above. For all genomic prediction analyses involving k-fold cross validation, we used GBLUP as previously described (Taylor 2014) and implemented in SVS v8.8.2 or v8.8.3 (Golden Helix), where the variance components were again estimated using the REML-based EMMA technique (Kang et al. 2010) with a genomic relationship matrix ðG) (VanRaden 2008; Taylor 2014). For WTD GBLUP analyses, consider the general mixed model equation: y ¼ Xf Bf þ u þ e, across n WTD samples where fixed effects specified as Bf include the intercept and any additional covariates (i.e., U.S. region, sex, age); but also assume VarðeÞ ¼ s2e I, as above, and that the random effects u are additive genetic merits (i.e., genomically estimated breeding values or GEBVs) for these WTD samples, which are produced from m markers as u ¼ Ma, where M is a n · m matrix, and a is a vector where ak is the allele substitution effect (ASE) for marker k: In this study, we used overall normalization for matrix M, as implemented in SVS v8.8.2 or v8.8.3 (Golden Helix), and explored solutions with and without gender corrections (i.e., full dosage compensation, no dosage compensation) (Taylor 2014), to produce GEBVs for all WTD samples as well as estimates of ASE for all SNPs. Moreover, considering that all training set samples precede the validation set, we define Z ¼ ½Ij0 , where the width and height of I is given as nt , the width of the zero matrix is given as nv , and the
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height of the zero matrix is nt . Thus we can partition u, Xf , and y ut according origin their (i.e., training vs. validation set) as u ¼ uv , to Xft y Xf ¼ , y ¼ t , and compute a genomic relationship matrix Xfv yv using all samples for use with the EMMA technique (Kang et al. 2010); to implement a mixed model for the training set as follows: yt ¼ Xft Bf þ Zu þ et , where VarðuÞ ¼ s2G G and VarðZuÞ ¼ s2G ZGZ ’ . Finally, we predict the validation set phenotypes as: ^f þ u ^v , from the intercept and any validation covariates ^yv ¼ Xfv B ^v . Additional formulae and Xfv as well as the predicted values of u supporting documentation are available at https://doc.goldenhelix.com/SVS/latest/svsmanual/mixedModelMethods/overview.html#gblupproblemstmt. Notably, prior to this study, GEBVs were not estimated or utilized in WTD, and thus they cannot be expected to be intuitive or easily understood by U.S. WTD farmers or relevant regulatory agencies. However, the predicted CWD binary phenotypes are both intuitive and easily understood as estimates of enhanced or reduced susceptibility to CWD. Because GBLUP predicts CWD binary phenotypes across a range of values (i.e., 0 to 1), SVS v8.8.2 and v8.8.3 (Golden Helix) considers predicted values of 0.5 and higher as “1”, and , 0.5 as “0”, thus facilitating the calculation of important summary statistics which require binary classifications. Justification for rounding is evident within the histograms representing the frequency distributions of the predicted CWD binary phenotypes (Fig. S1), the relevant GEBVs (Fig. S2), and the relationship between the predicted CWD binary phenotypes and the relevant GEBVs (Fig. S3); with an obvious break that occurs at 0.50 (Fig. S1-S3). Binary summary statistics for GBLUP-based genomic predictions with k-fold (k = 3; k = 5) cross validations (n = 50 iterations) were computed in SVS v8.8.2 or v8.8.3 (Golden Helix) as follows: Area Under the Curve as AUC ¼ nU1 n1 2 , where n1 is the sample size of observations with CWD binary phenotypes of 0, n2 is the sample size of observations with CWD binary phenotypes of 1, and U1 ¼ R1 2 n1 ðn21 þ1Þ, where R1 is the sum of the ranks for the predicted binary CWD phenotypes with actual phenotypes of 0 (from CWD diagnostics); Mathews Correlation CoTP TN 2 FP FN ffi, where TP is the efficient as MCC ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðTPþFNÞ ðFPþTNÞ ðTPþFPÞ ðFNþTNÞ
number of true CWD positives (from CWD diagnostics), TN is the number of true CWD non-detects (from CWD diagnostics), FP is the number of false positives, and FN is the number of false non-detects among the predicted CWD binary phenotypes; Genomic Prediction TPþTN ; Sensitivity (true positive rate) as Accuracy as ACC ¼ TPþFNþFPþTN TP TN TPR ¼ TPþFN ; Specificity (true negative rate) ffias SPC ¼ FPþTN ; Root rP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n 2 ðy 2^ y Þ i i i¼1 . For the GBLUP preMean Square Error as RMSE ¼ n dicted CWD Scores (0, 1, 2), we also produced and report relevant summary statistics from the k-fold (k = 3; k = 5) cross validations (n = 50 iterations) computed in SVS v8.8.2 or v8.8.3 (Golden Helix) as follows: Pn Pearson’s Product-Moment Correlation Coefficient as yÞ ðyi 2 yÞðy^i 2 ^ ry;^y ¼ i¼1ðn 2 1Þsy s^y where sy and s^y are the standard deviations; P Residual Sum ofPSquares as RSS ¼ ni¼1 ðyi 2^yÞ2 ; Total Sum of n 2 Squares TSS ¼ i¼1 ðyi 2 yÞ ; R-Squared as R2 ¼ 1 2 RSS TSS; Root qffiffiffiffiffiffi RSS Mean Square as n ; Mean Absolute Error as RMSE ¼ P Error MAE ¼ n1 ni¼1 yi 2 ^yi . Randomizing and blinding All GBLUP-based k-fold (k = 3; k = 5) cross validations (i.e., CWD binary; CWD-scores) were performed using automated random sampling to define the validation set (i.e., to predict on) and the training set, for the specified values of k.
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Figure 1 EMMAX binary case-control (0, 1) genome-wide association analyses (GWAA) for Chronic Wasting Disease (CWD) in farmed U.S. whitetailed deer (Odocoileus virginianus; hereafter WTD). All dual-panel manhattan plots depict -log10 P-values and the proportion of phenotypic variance explained (PVE) by white-tailed deer marker-effects on the y-axis, and the comparative position of all probe sequences on the x-axis, as inferred by blastn alignment with the bovine genome (ARS-UCD1.2). All analyses include diagnostically confirmed CWD positive (n = 284) and CWD non-detect (n = 523) WTD, and marker-based GRM heritability estimates (½h2 ¼ s2g = ðs2g þ s2e Þ ) (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). a, EMMAX GWAA for CWD with no fixed-effect covariates, high GRM heritability estimates (h2 ¼ 0:637 6 0:070) and relevant positional candidate genes. b, EMMAX GWAA for CWD with fixed-effect covariates (sex, age, U.S. region), high GRM heritability estimates (h2 ¼ 0:546 6 0:076Þ and relevant positional candidate genes. c, EMMAX GWAA for CWD with fixed-effect covariates (sex, age, U.S. region, CWD-scores), moderate GRM heritability estimates (h2 ¼ 0:337 6 0:079Þ and relevant positional candidate genes.
Data availability Accession codes are as follows: Illumina sequence data (SRA: SRR10313416-SRR10313421); genotype and phenotype data (DRYAD https://doi.org/10.5061/dryad.xd2547dcw).
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RESULTS AND DISCUSSION Using three reduced representation libraries (n = 374 farmed U.S. WTD) and Illumina paired-end sequencing for reference mapping and variant prediction, we successfully constructed a custom
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Affymetrix Axiom 200K SNP array for the WTD (See Methods). All probe sequences were aligned to the new PacBio long-read bovine genome assembly (ARS-UCD1.2; GCA_002263795.2), thus creating a comparative marker map. Thereafter, we genotyped a cohort of farmed WTD diagnostically classified (See Methods) as CWD positive (n = 284) and CWD non-detect (n = 523) from three U.S. geographic regions (Midwest, Northeast, South). Genome-wide association analyses were conducted using a mixed linear model with genomic relationship matrix and variance component analysis, thus yielding marker-based heritability estimates (GRM heritability), as implemented in EMMAX (Kang et al. 2010; Segura et al. 2012). All GWAA’s were carried out using 123,987 quality filtered SNPs for two dependent variables including a binary case-control variable (0 = non-detect; 1 = CWD positive) (Figure 1), and an interval variable (CWD-scores) which simultaneously reflects both the total number of CWD positive diagnostic tissues (i.e., 0, 1, 2) as well as the positive tissue types (i.e., 1 = lymph node only; 2 = lymph node and obex; Figure 2); with non-zero CWD-scores accurately modeling the natural progression of disease (Thomsen et al. 2012). Across all GWAA’s (Figure 1, Figure 2), GRM heritability estimates were
moderate to high (i.e., h2 ¼ 0:337 6 0:079 ─ 0:637 6 0:070); thus confirming that differential susceptibility to CWD in WTD is under genetic control, and that host genomic background also influences variation in disease progression. Herein we also confirm the PRNP gene as a major risk locus, and specifically, the codon 96 missense variant (G96S; binary case-control P-value , 6.30E-11; CWD-scores P-value , 1.49E-15) as well as one upstream promoter SNP (Affx-574071595; CWD-scores P-value # 5.40E-06) as being significantly associated with differential susceptibility to CWD, and with variation in natural disease progression among WTD (Figure 1, Figure 2, Table S1) (O’Rourke et al. 2004; Seabury et al. 2007). However, it should also be noted that 11 CWD-positive WTD possessed the codon 96SS genotype, and the proportion of phenotypic variance explained (PVE) by even the largest-effect PRNP SNPs detected across all GWAA’s (G96S, Promoter Affx-574071595) was , 0.11 (Figure 1, Figure 2), indicating that loci other than PRNP influence differential susceptibility and disease progression. These results are compatible with prior analyses performed in mice; where incubation times for transmissible spongiform encephalopathies were largely influenced by a genetic architecture independent of PRNP
Figure 2 EMMAX genome-wide association analyses (GWAA) for Chronic Wasting Disease (CWD) in farmed U.S. white-tailed deer (Odocoileus virginianus; hereafter WTD) using an interval variable (CWD-scores) to simultaneously reflect both the total number of CWD positive diagnostic tissues (i.e., 0, 1, 2) as well as the positive tissue types (i.e., 1 = lymph node only; 2 = lymph node and obex). All dual-panel manhattan plots depict -log10 P-values and the proportion of phenotypic variance explained (PVE) by white-tailed deer marker-effects on the y-axis, and the comparative position of all probe sequences on the x-axis, as inferred by blastn alignment with the bovine genome (ARS-UCD1.2). All analyses include diagnostically confirmed CWD positive (n = 284) and CWD non-detect (n = 523) WTD, and marker-based GRM heritability estimates (½h2 ¼ s2g = ðs2g þ s2e Þ ) (Kang et al. 2010; Segura et al. 2012; Seabury et al. 2017; Smith et al. 2019). a, EMMAX GWAA for CWD-scores with no fixed-effect covariates, high GRM heritability estimates (h2 ¼ 0:589 6 0:069) and relevant positional candidate genes. b, EMMAX GWAA for CWDscores with fixed-effect covariates (sex, age, U.S. region), high GRM heritability estimates (h2 ¼ 0:515 6 0:075Þ and relevant positional candidate genes.
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n■ Table 1 Summary of chronic wasting disease genomic predictions in farmed U.S. white-tailed deer (Odocoileus virginianus) k-Fold Subsample k k k k
= = = =
3 3 5 5
GBLUP Model Covariates
Mean AUC (SD)a
None 0.8471 (0.0068) Sex, Age, U.S. Region 0.8534 (0.0063) None 0.8485 (0.0053) Sex, Age, U.S. Region 0.8542 (0.0047)
Mean Mean Genomic Matthews Prediction Coefficient (SD) Accuracy (SD) 0.5871 0.5787 0.5940 0.5870
(0.0152) (0.0158) (0.0132) (0.0123)
0.8167 0.8119 0.8198 0.8159
(0.0066) (0.0070) (0.0057) (0.0055)
Mean Sensitivity (SD) 0.6447 0.6735 0.6496 0.6716
Mean Specificity (SD)
(0.0117) (0.0122) (0.0110) (0.0110)
0.9101 0.8870 0.9121 0.8942
(0.0091) (0.0081) (0.0060) (0.0066)
Mean RMSE (SD)b 0.3768 0.3746 0.3754 0.3734
(0.0043) (0.0040) (0.0028) (0.0025)
a Area Under the Curve (AUC; Wilcoxon Mann Whitney Method; See Methods). b
Root Mean Square Error (RMSE; See Methods).
(Iyegbe et al. 2010). Across all GWAA’s (n = 123,987 SNPs), only 61 SNPs met a nominal significance level for polygenic traits (Pvalue # 5E-05) (Wellcome Trust Case Control Consortium 2007; Seabury et al. 2017), with 17 detected in more than one GWAA. This is relatively unsurprising since EMMAX is known to produce conservative P-values; particularly when large-effect regions exist (Zhou and Stephens 2012). Moreover, the architecture of both investigated CWD traits (Figure 1, Figure 2) is such that few large-effect regions exist (i.e., PVE $ 0.03); but together with many smaller-effect regions, a significant proportion of the phenotypic variance can be explained. Interestingly, an investigation of all GWAA’s revealed many of the same positional candidate genes (Figure 1, Figure 2; PVE $ 0.02); the majority of which have been implicated in the pathophysiology of various prion diseases including scrapie (i.e., ACSL4, CA3, KLF6), bovine spongiform encephalopathy (i.e., NPAS3, BACH2, EPHA7, ITGA4), spontaneous and familial Creutzfeldt-Jakob disease (i.e., HIST1H4D/OPCML, LAMA3, TTC7B), and various other neurodegenerative conditions including Alzheimer’s (i.e., DGKI, SFRP1, SLC24A4) and Parkinson’s disease (BCL11B) (Scherzer et al. 2007; Tang et al. 2009; Tortosa et al. 2011; Silver et al. 2012; Cohen et al. 2013; Lambert et al. 2013; Filali et al. 2014; Lee et al. 2014; Kipkorir et al. 2015; Xerxa et al. 2016; Esteve et al. 2019; Majer et al. 2019). Summary data for all GWAA’s and positional candidate genes (Figure 1, Figure 2) as well as the corresponding P-P plots are provided in the supplementary information (Table S1; Additional Files 1-5 in DRYAD). To investigate the potential for developing a genomically-estimated CWD eradication program for farmed WTD, we used genomic best linear unbiased prediction (GBLUP) in conjunction with k-fold cross validation and random sampling (Taylor 2014). Briefly, WTD data (i.e., genotypes, CWD diagnostic phenotypes, and other metadata) were randomly partitioned into k -subsamples (k = 3, k = 5), and one of these subsamples was then selected (i.e., within a discrete fold) to serve as the validation set for genomic prediction; thus the GBLUP model was fit using the remaining (i.e., k -1) subsamples within that fold (i.e., the training data); until all subsamples were used for both training and prediction. All cross validations with random sampling were run for 50 iterations, with each iteration consisting of either three or five folds (k = 3, k = 5), and summary statistics were produced (See Table 1; Methods; Additional Files 6-29 in DRYAD). Binary GBLUP models fit with no fixed-effect covariates (k = 3, k = 5) produced high mean genomic prediction accuracies ($ 0.8167) and specificities ($ 0.9101), with small standard deviations, but lower mean sensitivities (# 0.6496), indicating that false negatives pose the greatest challenge for reducing the prevalence of CWD via genomic prediction (Table 1; See Methods). Similar results were also obtained when binary GBLUP models were fit using sex, age, and U. S. region of origin as fixed-effect covariates (k = 3, k = 5; Table 1). However, in addition to false negatives, some false positive genomic predictions were also observed; most likely due to underlying genomic susceptibility coupled with either very early stages of disease (i.e., CWD
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non-detect diagnostically) and/or differences in exposure (Tsairidou et al. 2014). All results were robust to the inclusion or exclusion of non-autosomal loci (i.e., X, MT; 123,987 SNPs vs. 120,808 SNPs, respectively), and to full dosage compensation vs. no dosage compensation when putative X-linked SNPs were included (Table 1; Additional Files 6-29 in DRYAD). GBLUP models fit with the CWD-scores (0, 1, 2), thus reflecting the natural progression of disease, produced lower mean genomic prediction accuracies (i.e., # 0.6007; See Methods; DRYAD), regardless of the inclusion or exclusion of fixed-effect covariates (sex, age, U.S. region), nonautosomal loci, or the implementation of full dosage compensation vs. no dosage compensation (k = 3, k = 5). CONCLUSIONS Herein, we demonstrate that differential susceptibility to CWD and variation in natural disease progression are both heritable, polygenic traits in farmed U.S. WTD, and that genome-wide SNP data can be used to produce accurate genomic predictions for risk ($ 0.8167); thereby providing the first novel strategy for reducing the prevalence of CWD. Moreover, given the genomic architecture of these traits, we also demonstrate that PRNP genotyping alone cannot be expected to facilitate an eradication program, or to rapidly reduce the overall prevalence of CWD in farmed U.S. WTD. AUTHOR CONTRIBUTIONS C.M.S designed the research with input from T.A.N. M.A.L. and T.A.N. provided CWD diagnostic data, other relevant animal metadata, and biological samples for DNA isolation from existing agency repositories. C.M.S. and D. L. isolated and quantified DNA with assistance from P.F. E.K.B. prepared hair cards and quantified DNA. R.P.M. and C.D.J. prepared and sequenced reduced representation libraries. C.M.S. performed trimming, reference mapping, and variant prediction. C.M.S. designed the Affymetrix Axiom 200K SNP array with assistance from D.L.O. D.L.O. performed blastn searches to create comparative marker maps for the array. D.L.O. submitted reads to NCBI SRA. C.M.S. performed quality filtering of genotypes, genome wide association analyses with marker-based heritability estimates, alignment with positional candidate genes, and genomic predictions with k-fold cross validation and summary statistics. C.M.S. wrote the manuscript; incorporating input from D.L.O., E.K.B., D.L., P.F., R.P.M., C.D.J., M.A.L., and T.A.N. All authors edited and approved the manuscript. ACKNOWLEDGMENTS This material was made possible, in part, by a Cooperative Agreement from the United States Department of Agriculture’s Animal and Plant Health Inspection Service (APHIS). It may not necessarily express APHIS’ views. CMS acknowledges this support from USDA-MISAnimal and Plant Health Inspection Service (grant AP17VSSPRS00C126)
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» CWD RESEARCH « CONTINUED FROM PREVIOUS PAGE as well as support from Texas Parks and Wildlife Department (grant 475613) and a charitable gift from the American Chronic Wasting Disease Foundation. LITERATURE CITED Bistaffa, E., T. T. Vuong, F. A. Cazzaniga, L. Tran, G. Salzano et al., 2019 Use of different RT-QuIC substrates for detecting CWD prions in the brain of Norwegian cervids. Sci. Rep. 9: 18595. https://doi.org/10.1038/s41598-01955078-x Cohen, E., D. Avrahami, K. Frid, T. Canello, E. Levy Lahad et al., 2013 Snord 3A: A molecular marker and modulator of prion disease progression. PLoS One 8: e54433. https://doi.org/10.1371/journal.pone.0054433 Edmunds, D. R., M. J. Kauffman, B. A. Schumaker, F. G. Lindzey, W. E. Cook et al., 2016 Chronic Wasting Disease Drives Population Decline of White-tailed Deer. PLoS One 11: e0161127. https://doi.org/10.1371/ journal.pone.0161127 Esteve, P., J. Rueda-Carrasco, M. Inés Mateo, M. J. Martin-Bermejo, J. Draffin et al., 2019 Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer’s disease pathogenesis. Nat. Neurosci. 22: 1258–1268. https://doi.org/10.1038/s41593-019-0432-1 Filali, H. I., Martín-Burriel I., Harders F., Varona L., Hedman C. et al., 2014 Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie. BMC Genomics 15: 59. https://doi.org/10.1186/1471-2164-15-59 Gavin, C., D. Henderson, S. L. Benestad, M. Simmons, and A. Adkin, 2019 Estimating the amount of Chronic Wasting Disease infectivity passing through abattoirs and field slaughter. Prev. Vet. Med. 166: 28–38. https://doi.org/10.1016/j.prevetmed.2019.02.016 Grear, D. A., M. D. Samuel, J. A. Langenberg, and D. Keane, 2006 Demographic patterns and harvest vulnerability of chronic wasting disease infected white-tailed deer in Wisconsin. J. Wildl. Manage. 70: 546– 553. https://doi.org/10.2193/0022-541X(2006)70[546:DPAHVO]2.0.CO;2 Halley, Y. A., D. L. Oldeschulte, E. K. Bhattarai, J. Hill, R. P. Metz et al., 2015 Northern Bobwhite (Colinus virginianus) Mitochondrial Population Genomics Reveals Structure, Divergence, and Evidence for Heteroplasmy. PLoS One 10: e0144913. https://doi.org/10.1371/ journal.pone.0144913 Halley, Y. A., S. E. Dowd, J. E. Decker, P. M. Seabury, E. Bhattarai et al., 2014 A draft de novo genome assembly for the northern bobwhite (Colinus virginianus) reveals evidence for a rapid decline in effective population size beginning in the Late Pleistocene. PLoS One 9: e90240. https://doi.org/10.1371/journal.pone.0090240 Iyegbe, C. O., O. O. Abiola, C. Towlson, J. F. Powell, and S. A. Whatley, 2010 Evidence for Varied Aetiologies Regulating the Transmission of Prion Disease: Implications for Understanding the Heritable Basis of Prion Incubation Times. PLoS One 5: e14186. https://doi.org/10.1371/ journal.pone.0014186 Kang, H. M., J. H. Sul, S. K. Service, N. A. Zaitlen, S. Y. Kong et al., 2010 Variance component model to account for sample structure in genome-wide association studies. Nat. Genet. 42: 348–354. https://doi.org/ 10.1038/ng.548 Kipkorir, T., C. M. Colangelo, and L. Manuelidis, 2015 Proteomic analysis of host brain components that bind to infectious particles in CreutzfeldtJakob disease. Proteomics 15: 2983–2998. https://doi.org/10.1002/ pmic.201500059 Lambert, J. C., C. A. Ibrahim-Verbaas, D. Harold, A. C. Naj, R. Sims et al., 2013 Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease. Nat. Genet. 45: 1452–1458. https://doi.org/ 10.1038/ng.2802 Lee, S. M., M. Chung, K. J. Hwang, Y. R. Ju, J. W. Hyeon et al., 2014 Biological network inferences for a protection mechanism against familial Creutzfeldt-Jakob disease with E200K pathogenic mutation. BMC Med. Genomics 7: 52. https://doi.org/10.1186/1755-8794-7-52 Majer, A., S. J. Medina, D. Sorensen, M. J. Martin, K. L. Frost et al., 2019 The cell type resolved mouse transcriptome in neuron-enriched brain tissues from the hippocampus and cerebellum during prion disease. Sci. Rep. 9: 1099. https://doi.org/10.1038/s41598-018-37715-z VanRaden, P. M., 2008 Efficient methods to compute genomic predictions. J. Dairy Sci. 91: 4414–4423. https://doi.org/10.3168/jds.2007-0980 Wellcome Trust Case Control Consortium, 2007 Genome-wide association 1 study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447: 661–678. https://doi.org/10.1038/nature05911 Williams, E. S., and S. Young, 1980 Chronic wasting disease of captive mule deer: A spongiform encephalopathy. J. Wildl. Dis. 16: 89–98. https:// doi.org/10.7589/0090-3558-16.1.89
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Moreno, J. A., and G. C. Telling, 2018 Molecular Mechanisms of Chronic Wasting Disease Prion Propagation. Cold Spring Harb. Perspect. Med. 8. https://doi.org/10.1101/cshperspect.a024448 O’Rourke, K. I., T. R. Spraker, L. K. Hamburg, T. E. Besser, K. A. Brayton et al., 2004 Polymorphisms in the prion precursor functional gene but not the pseudogene are associated with susceptibility to chronic wasting disease in whitetailed deer. J. Gen. Virol. 85: 1339–1346. https://doi.org/10.1099/vir.0.79785-0 Oldeschulte, D. L., Y. A. Halley, M. L. Wilson, E. K. Bhattarai, W. Brashear et al., 2017 Annotated Draft Genome Assemblies for the Northern Bobwhite (Colinus virginianus) and the Scaled Quail (Callipepla squamata) Reveal Disparate Estimates of Modern Genome Diversity and Historic Effective Population Size. G3 (Bethesda) 7: 3047–3058. https:// doi.org/10.1534/g3.117.043083 Osterholm, M. T., C. J. Anderson, M. D. Zabel, J. M. Scheftel, K. A. Moore et al., 2019 Chronic Wasting Disease in Cervids: Implications for Prion Transmission to Humans and Other Animal Species. MBio 10. https:// doi.org/10.1128/mBio.01091-19 Scherzer, C. R., A. C. Eklund, L. J. Morse, Z. Liao, J. J. Locascio et al., 2007 Molecular markers of early Parkinson’s disease based on gene expression in blood. Proc. Natl. Acad. Sci. USA 104: 955–960. https:// doi.org/10.1073/pnas.0610204104 Seabury, C. M., C. A. Gill, J. W. Templeton, J. B. Dyar, J. N. Derr et al., 2007 Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter. J. Hered. 98: 678–686. https:// doi.org/10.1093/jhered/esm091 Seabury, C. M., D. L. Oldeschulte, M. Saatchi, J. E. Beever, J. E. Decker et al., 2017 Genome-wide association study for feed efficiency and growth traits in U.S. beef cattle. BMC Genomics 18: 386. https://doi.org/10.1186/ s12864-017-3754-y Seabury, C. M., E. K. Bhattarai, J. F. Taylor, G. G. Viswanathan, S. M. Cooper et al., 2011 Genome-wide polymorphism and comparative analyses in the whitetailed deer (Odocoileus virginianus): a model for conservation genomics. PLoS One 6: e15811. https://doi.org/10.1371/journal.pone.0015811 Segura, V., B. J. Vilhjálmsson, A. Platt, A. Korte, Ü. Seren et al., 2012 An efficient multi-locus mixed-model approach for genome-wide association studies in structured populations. Nat. Genet. 44: 825–830. https://doi.org/ 10.1038/ng.2314 Silver, M., E. Janousova, X. Hua, P. M. Thompson, G. Montana et al., 2012 Identification of gene pathways implicated in Alzheimer’s disease using longitudinal imaging phenotypes with sparse regression. Neuroimage 63: 1681–1694. https://doi.org/10.1016/j.neuroimage.2012.08.002 Smith, J. L., M. L. Wilson, S. M. Nilson, T. N. Rowan, D. L. Oldeschulte et al., 2019 Genome-wide association and genotype by environment interactions for growth traits in U.S. Gelbvieh cattle. BMC Genomics 20: 926. https://doi.org/10.1186/s12864-019-6231-y Sollars, E. S., A. L. Harper, L. J. Kelly, C. M. Sambles, R. H. Ramirez-Gonzalez et al., 2017 Genome sequence and genetic diversity of European ash trees. Nature 541: 212–216. https://doi.org/10.1038/nature20786 Tang, Y., W. Xiang, S. A. Hawkins, H. A. Kretzschmar, and O. Windl, 2009 Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity. J. Virol. 83: 9464–9473. https://doi.org/10.1128/JVI.00352-09 Taylor, J. F., 2014 Implementation and accuracy of genomic selection. Aquaculture 420–421: S8–S14. https://doi.org/10.1016/j.aquaculture.2013.02.017 Thomsen, B. V., D. A. Schneider, K. I. O’Rourke, T. Gidlewski, J. McLane et al., 2012 Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America: Effects of age, sex, polymorphism at PRNP codon 96, and disease progression. J. Vet. Diagn. Invest. 24: 878–887. https://doi.org/ 10.1177/1040638712453582 Tortosa, R., X. Castells, E. Vidal, C. Costa, M. Ruiz de Villa et al., 2011 2011 Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy. Vet. Res. (Faisalabad) 42: 109. https://doi.org/10.1186/1297-9716-42-109 Tsairidou, S., J. A. Wooliams, A. R. Allen, R. A. Skuce, S. H. McBride et al., 2014 Genomic Prediction for Tuberculosis Resistance in Dairy Cattle. PLoS One 9: e96728. https://doi.org/10.1371/journal.pone.0096728 Xerxa, E., M. Barbisin, M. N. Chieppa, H. Krmac, E. Vallino Costassa et al., 2016 Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy. PLoS One 11: e0153425. https://doi.org/10.1371/journal.pone.0153425 Zhou, X., and M. Stephens, 2012 Genome-wide efficient mixed-model analysis for association studies. Nat. Genet. 44: 821–824. https://doi.org/10.1038/ng.2310
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Th he Process of Syndication Step One: Whitetail Buck Syndicatio on • Defined Number of Shares • Shares Can Be Bought//Sold Sold & Resold • Each Share entitled to A Limited amount of Sem S en Annua ally
Step Two: Syndic cation Management e • Bo Booth ooth Exposure
• Co-op Advertissing
• So ocial Media
• Mating Certific cates
• Web e Exposure
• Managing Sem men Distribution
• Se emen Releases
• Offspring Exposure
• 1 Shareholder Fee Annually
Step Three: e Semen Disstribution ee • Semen Releases (30 Straws Annually) • Semen Released Annually for o Each Share Structure • All Semen Released, must b be used that year (30 straws)
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» FALLOW RESEARCH
FAILURE OF
FALLOW DEER TO DEVELOP CWD WHEN EXPOSED TO A
CONTAMINATED ENVIRONMENT
& INFECTED MULE DEER
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Jack C. Rhyan,1,6 Michael W. Miller,2 Terry R. Spraker,3 Matt McCollum,1 Pauline Nol,1 Lisa L. Wolfe,2 Tracy R. Davis,2 Lynn Creekmore,4 and Katherine I. O’Rourke5 1 National Wildlife Research Center, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Fort Collins, Colorado 80521, USA; 2 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, Colorado 80526-2097, USA; 3 Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, Colorado 80523, USA; 4 Natural Resources Research Center, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Fort Collins, Colorado 80526, USA; 5 US Department of Agriculture, Agricultural Research Service, 337 Bustad Hall, Washington State University, Pullman, Washington 99163, USA; 6 Corresponding author (email: jack.c.rhyan@aphis.usda.gov)
vus elaphus elaphus), moose (Alces alces), and, in preliminary studies, muntjac deer (Muntiacus reevesi; Williams and Young, 1980, 1982; Spraker et al., 1997; Kreeger et al., 2006; Martin et al., 2009; A. Young, pers. comm.). The CWD agent can be transmitted from infected animals and from environments contaminated with the excrement or carcasses of infected animals (Miller and Williams, 2003; Miller et al., 2004; Tamgüney et al., 2009). Intracerebral inoculation with brain suspension from CWD-infected whitetailed deer and elk resulted in progressive clinical disease in four of 13 fallow deer (Dama dama) between 24 and 37 mo postinoculation (mpi; Hamir et al., 2008). The four affected fallow deer, all of which had received disease-associated prion protein (PrPd) from white-tailed deer (WTD), and another clinically normal individual killed 26 mpi had small amounts of the abnormal PrPd in central nervous system (CNS) tissues but lacked spongiform degeneration in the neuropil and PrPd in lymphoid tissues (Hamir et al., 2008). At 4 yr postinoculation, the remaining inoculated fallow deer (one had received PrPd from WTD and 4 PrPd from elk) were alive and apparently healthy; however, between 51 and 60 mo all five became sick and were euthanized. On postmortem examination, all deer had spongiform encephalopathy and evidence of PrPd in CNS tissues (Hamir et al., 2011). We monitored a group of fallow deer and their offspring for evidence of prion infection
We monitored a herd of fallow deer (Dama dama) for evidence of prion infection for 7 yr by periodic postmortem examination of animals from the herd. The fallow deer were exposed to the chronic wasting disease (CWD) agent from mule deer by living in a paddock considered contaminated with infectivity from its history of housing CWD infected deer and, after the first year of the study, by comingling with infected mule deer (Odocoileus hemionus). At least 8 of 12 mule deer serving as sentinels for prion transmission and 25 additional mule deer serving as sources of infectivity developed clinical CWD or were otherwise confirmed to be infected with CWD via lymphoid tissue immunohistochemistry (IHC). In contrast, none of the 41 exposed fallow deer showed clinical signs suggestive of CWD, IHC staining of disease-associated prion in lymphoid or brain tissues, or evidence of spongiform degeneration in sections of brain stem at the level of the obex when sampled 18 mo to 7 yr after entering the mule deer paddock. The absence of clinical disease and negative IHC results in fallow deer housed in the same contaminated paddock for up to 7 yr and almost continuously exposed to CWDinfected mule deer for up to 6 yr suggests a species barrier or other form of resistance preventing fallow deer infection by the CWD agent or delaying progression of the disease in this species. Key words: Chronic wasting disease (CWD), Dama dama, fallow deer, prion, spongiform encephalopathy. ABSTRACT:
Chronic wasting disease (CWD) is caused by a prion strain (or strains) that infects several species of cervids including mule deer (Odocoileus hemionus), whitetailed deer (Odocoileus virginianus), elk (Cervus elaphus nelsoni), red deer (Cer7
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for up to 7 yr of exposure to CWD through living in a paddock contaminated by previous CWD studies and inhabited by infected mule deer. Our study was conducted at the Colorado Division of Wildlife’s (CDOW) Foothills Wildlife Research Facility (FWRF; Fort Collins, Colorado) between October 2000 and November 2007 under study protocols approved by the CDOW Animal Care and Use Committee (file 12– 2000). In October 2000, 22 female and 3 male, 6-mo-old fallow deer were purchased from a private herd, in a state in which CWD had not been diagnosed, and transported to the FWRF (Fig. 1). The private herd conducted routine slaughter surveillance for CWD and had never had an animal positive. We placed fallow deer in a ,2.0 ha paddock (‘‘Paddock C’’ in Fig. 2A of Miller and Wild, 2004) that was considered contaminated with the CWD agent because it had recently housed CWD-infected mule deer, contained fecal and urine residues from previous CWD studies (Miller and Williams, 2003; Miller and Wild, 2004; Miller et al., 2006), and was adjacent to paddocks in which a CWD-mule deer study was ongoing. Between June 11 and 25 August 2002, 18 fallow deer fawns (12 males, 6 females) were born to the original study animals in the contaminated paddock (Fig. 1); two died as neonates. The three original male fallow deer were subsequently vasectomized, and males born in the paddock were euthanized and necropsied in the first 2 yr of life to preclude birth of additional fawns. During the study, we added mule deer to the paddock holding the fallow deer (Table 1). Twelve of these mule deer were #151 days old and were not known to be exposed to CWD (Fig. 1). Nine of the 12 had been born to dams in the FWRF, and three were raised as orphans by individuals elsewhere in Colorado. These deer served as sentinels for CWD transmission in the paddock and are called ‘‘sentinel mule deer’’ hereafter. Another 27 mule
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FIGURE 1. Temporal relationships between the addition and removal of fallow deer (Dama dama; n541) and ‘‘sentinel’’ (n512) and ‘‘infection-source’’ (n525) mule deer (Odocoileus hemionus) in a study of fallow deer susceptibility to chronic wasting disease. None of the fallow deer showed evidence of prion infection when removed at time points 18– 84 mo after the start of the study. Infections were confirmed in eight of the sentinel mule deer (solid circles); two sentinels appeared to be uninfected at postmortem examination (open circles), and the infection status of two others (gray centers) were not determined. For infection source mule deer, the gray bars represent the number of potentially infected deer days (number of deer 3 number of days) of exposure for each month where at least one infection-source deer was present in the paddock.
deer placed in the paddock were $352 days old and had been exposed to the CWD agent during other studies prior to their placement in the fallow deer study paddock; 16 of these were positive on tonsil biopsy immunohistochemistry (IHC; Wolfe et al., 2002) at the time they were added to the fallow deer paddock. In all, 25 of these animals, hereafter called ‘‘infection-source mule deer,’’ were shown to be infected and served as additional sources of infection. Two of the 27 exposed deer were placed in the paddock 2.5 and 15.5 mo prior to the end of the study. These two were negative at the end of the study and are not included in the infection-source mule deer. Syntopic fallow deer and mule deer used the same automatic water source, shelters, pasture space, and feed bunks but rarely socialized
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TABLE 1. Numbers of sentinel and infection-source mule deer (Odocoileus hemionus) and fallow deer (Dama dama) added to and removed from a prion-contaminated paddock during a study of fallow deer susceptibility to chronic wasting disease (CWD). Values in parentheses are the numbers of deer developing clinical signs of or testing positive for CWD. Study year 2000–01
a
2001–02
2002–03
2003–04
2004–05
2005–06
2006–07
Total
Mule deer added Sentinel Infection source
4 0
6 1
2 (0) 0
1 (0) 1 (1)
0 12
2 9
0 1
0 1
0 1
0 4 (4)
2 (2) 1 (1)
0 2 (2)
12 25
Mule deer removed Sentinel Infection source
3 (2) 4 (4)
4 (4) 13 (13)
12b (8) 25 (25)
Fallow deer added Original animals 2002 birth cohort Fallow deer removed Original animals 2002 birth cohort
25 0 0 0
0 16c 5 (0) 0b (0)
0 0
0 0
0 0
0 0
0 0
25 16
6 (0) 8 (0)
8 (0) 4 (0)
0 0
0 0
6 (0) 4 (0)
25 (0) 16 (0)
a
Years are measured from November to October (e.g., November 2000 to October 2001), except that 2000–01 included part of October 2000 and 2006–07 included part of November 2007.
b
Two of the 12 developed clinical illness but laboratory testing was not done; two were euthanized at 7 mo of age and were PrPd negative in lymphoid tissue.
c
Two neonatal deaths not included in count.
or made direct contact between species while grazing. All deer were observed daily for clinical signs of CWD, and mule deer were routinely euthanized after clinical signs of CWD were observed. Mule deer were necropsied, and their retropharygeal lymph nodes collected and tested by an enzyme-linked immunosorbent assay (Hibler et al., 2003) and IHC (Spraker et al., 2002), except in 10 cases that had previously tested positive by tonsil biopsy IHC. In all, 8 of 12 sentinel mule deer and all 25 infection-source mule deer were confirmed as infected with CWD via lymphoid tissue IHC over the 7-yr study (Table 1). Of the four sentinel mule deer that were not confirmed as infected, two were euthanized at 7 mo of age due to chronic diarrhea and were negative for PrPd in lymphoid tissue, and two were euthanized due to illness but laboratory specimens were lost prior to testing for prion infection (Fig. 1). Of the eight
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sentinel mule deer with laboratory evidence of CWD and the 25 infectionsource mule deer, three died of immobilization complications, two of CWD, and the rest were euthanized following development of clinical signs consistent with CWD. In contrast to mule deer, none of the 41 exposed fallow deer showed clinical signs suggestive of CWD. Fallow deer were euthanized and necropsied at various times during the study (Table 1). Tissues collected for IHC staining for PrP d included brain (obex at the level of the dorsal motor nucleus of the vagus), palatine tonsil, and medial retropharyngeal lymph node. In selected animals, pharyngeal tonsil (n514), ileum (n519), jejunum (n517), mesenteric lymph node (n513), and rectum (n58) also were examined by IHC for PrPd. IHC staining of PrPd was not detected in any of the tissue sections from fallow deer. No evidence of spongiform degeneration was
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found in any of the brain sections from fallow deer. We extracted DNA from liver specimens from 23 fallow deer, and the open reading frame of the PRNP gene was amplified and sequenced as described by Spraker et al. (2004). Sequences were identical to that reported in GenBank accession AY286007 except for a single synonymous change (aac/aat, encoding asparagine) at codon 138. Fourteen of these fallow deer were heterozygous for the polymorphism, six were homozygous for the aat variant, and three were homozygous for the aac variant. Susceptibility to some prion diseases is associated with single amino acid substitutions in the prion protein. Further, the species barrier, which limits the transmission of prion strains between species (Pattison and Jones, 1968; Prusiner, 1998), also may be linked to single amino acid mismatches between the infected donor animal and the naive recipient. In this experiment, the prion protein amino acid differences between the putative infected donor (mule deer) and the naive recipient (fallow deer) are limited to two residues. The mule deer PRNP gene encodes serine at codon 138 (138S), and the fallow deer prion gene encodes asparagine (138N). The mule deer gene encodes glutamine at codon 226 (226Q), and the fallow deer prion gene encodes glutamic acid at that site (226E). The substitution at codon 226 had no effect on susceptibility to experimental oral challenge of red deer with CWD-infected elk brain (Balachandran et al., 2009), but the effect of this mismatch between mule deer and fallow deer is unknown. The extent of variability in the PRNP gene of fallow deer in the USA is not known. The fallow deer in this study had a single PRNP genotype; no other alleles of the PRNP gene in fallow deer have been reported in the small numbers examined (this publication; GenBank accessions EF165089, EF139175, AY639094, AY286007; K. I. O’Rourke, unpubl. data on farmed US fallow deer).
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If other alleles of the gene are prevalent in farmed US fallow deer, additional in vitro or in vivo studies on their effect on susceptibility are warranted. Eight of 10 sentinel mule deer maintained in the fallow deer paddock for $530 days—including two individuals born in the paddock—were positive for CWD, indicating that transmission of the agent was occurring throughout most if not all of the study period (Fig. 1). The absence of clinical disease and negative IHC results in fallow deer housed in a contaminated paddock for up to 7 yr with 6 yr of essentially continuous exposure to infection source and infected sentinel mule deer (Fig. 1) suggests that a species barrier or other form of resistance exists that either prevented fallow deer from becoming infected by the CWD agent or delayed progression of the disease such that infections could not be demonstrated. These findings are consistent with the absence of reported field cases of CWD in fallow deer, and with results of a recent intracerebral inoculation study using CWD agent from WTD and elk (Hamir et al., 2008). Those authors suggested that it may not be possible to transmit CWD to fallow deer by more natural routes and that a relatively strong species barrier against CWD infection may exist for fallow deer. The eventual development of spongiform encephalopathy with PrPd amplification in the deer 51 to 60 mpi is indicative of a delayed disease progression following intracerebral inoculation as compared with other species. Suffolk sheep developed spongioform change and clinical illness and demonstrated PrPd amplification as early as 36 mo postintracerebral inoculation with PrPd of mule deer origin (Hamir et al., 2006). Whether fallow deer are completely resistant to developing CWD following natural exposure or experience greatly delayed disease progression is unknown and may warrant further study. Our study was funded by the US Department of Agriculture (USDA), Ani-
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mal and Plant Health Inspection Service, Veterinary Services, and the Colorado Division of Wildlife, with some funding also provided by the USDA, Agriculture Research Service. We thank Tom Gidlewski, Karl Held, and Roger Thompson for assistance with necropsies. Tom Gidlewski provided helpful comments on an earlier draft of our manuscript. LITERATURE CITED BALACHANDRAN, A., N. P. HARRINGTON, J. ALGIRE, A. SOUTYRINE, T. R. SPRAKER, M. JEFFREY, L. GONZALEZ, AND K. I. O’ROURKE. 2010. Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): Early detection and late stage distribution of protease-resistant prion protein. Canadian Veterinary Journal 51: 169–178. HAMIR, A. N., J. J. GREENLEE, E. M. NICHOLSON, R. A. KUNKLE, J. A. RICHT, J. M. MILLER, AND S. M. HALL. 2011. Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: Final report. Canadian Journal of Veterinary Research 75: 152–156. ———, R. A. KUNKLE, R. C. CUTLIP, J. M. MILLER, E. S. WILLIAMS, AND J. A. RICHT. 2006. Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation. Journal of Veterinary Diagnostic Investigation 18: 558–565. ———, ———, E. M. NICHOLSON, J. M. MILLER, S. M. HALL, H. SCHOENENBRUECHER, B. W. BRUNELLE, AND J. A. RICHT. 2008. Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer. Journal of Comparative Pathology 138: 121–130. HIBLER, C. P., K. L. WILSON, T. R. SPRAKER, M. W. MILLER, R. R. ZINK, L. L. DEBUSE, E. ANDERSEN, D. SCHWEITZER, J. A. KENNEDY, L. A. BAETEN, J. F. SMELTZER, M. D. SALMAN, AND B. E. POWERS. 2003. Field validation and assessment of an enzyme-linked immunosorbent assay for detecting chronic wasting disease in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni). Journal of Veterinary Diagnostic Investigation 15: 311–319. KREEGER, T. J., D. L. MONTGOMERY, J. E. JEWELL, W. SCHULZ, AND E. S. WILLIAMS. 2006. Oral transmission of chronic wasting disease in captive Shira’s moose. Journal of Wildlife Diseases 42: 640–645. MARTIN, S., M. JEFFREY, L. GONZALEZ, S. SISO, H. W. REID, P. STEELE, M. P. DAGLEISH, M. J. STACK,
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M. J. CHAPLIN, AND A. BALACHANDRAN. 2009. Immunohistochemical and biochemical characteristics of BSE and CWD in experimentally infected European red deer (Cervus elaphus elaphus). BMC Veterinary Research 5: 26. doi:10.1186/1746-6148-5-26. MILLER, M. W., N. T. HOBBS, AND S. J. TAVENER. 2006. Dynamics of prion disease transmission in mule deer. Ecological Applications 16: 2208– 2214. ———, AND M. A. WILD. 2004. Epidemiology of chronic wasting disease in captive white-tailed and mule deer. Journal of Wildlife Diseases 40: 320–327. ———, AND E. S. WILLIAMS. 2003. Horizontal prion transmission in mule deer. Nature 425: 35–36. ———, ———, N. T. HOBBS, AND L. L. WOLFE. 2004. Environmental sources of prion transmission in mule deer. Emerging Infectious Diseases 10: 1003–1006. PATTISON, I. H., AND K. M. JONES. 1968. Modification of a strain of mouse-adapted scrapie by passage through rats. Research in Veterinary Science 9: 408–410. PRUSINER, S. B. 1998. Prions. Proceedings of the National Academy of Sciences USA 95: 13363– 13383. SPRAKER, T. R., A. BALACHANDRAN, D. ZHUANG, AND K. I. O’ROURKE. 2004. Variable patterns of distribution of PrPCWD in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease. Veterinary Record 155: 295–302. ———, M. W. MILLER, E. S. WILLIAMS, D. M. GETZY, W. J. ADRIAN, G. G. SCHOONVELD, R. A. SPOWART, K. I. O’ROURKE, J. M. MILLER, AND P. A. MERZ. 1997. Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado. Journal of Wildlife Diseases 33: 1–6. ———, K. I. O’ROURKE, A. BALACHANDRAN, R. R. ZINK, B. A. CUMMINGS, M. W. MILLER, AND B. E. POWERS. 2002. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease. Journal of Veterinary Diagnostic Investigation 14: 3–7. TAMGÜNEY, G., M. W. MILLER, L. L. WOLFE, T. M. SIROCHMAN, D. V. GLIDDEN, C. PALMER, A. LEMUS, S. J. DEARMOND, AND S. B. PRUSINER. 2009. Asymptomatic deer excrete infectious prions in faeces. Nature 461: 529–532. WILLIAMS, E. S., AND S. YOUNG. 1980. Chronic wasting disease of captive mule deer: A spongiform encephalopathy. Journal of Wildlife Diseases 16: 89–98. ———, AND ———. 1982. Spongiform encephalopathy of Rocky Mountain elk. Journal of Wildlife Diseases 18: 465–471. u
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» DEER URINE
Ensuring the Safety of Natural Deer Urine Products By Davin Henderson CWD Evolution Chronic wasting disease (CWD) has affected many facets of the hunting and deer farming world. Throughout the last 5 years many state wildlife agencies have sought out measures to prevent the spread of CWD to their states. To that end many states across the country have either attempted to ban or banned the use of natural urine products. The banning of natural deer urine products has had a direct impact to both retailers in the hunting industry and deer farming producers. Despite thin evidence that deer urine can spread CWD an increasing number of states have enacted or are considering bans. To address the safety of deer urine products the major manufacturers sought out advice from wildlife agency experts and leading scientists to
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create a series of measures that go above and beyond USDA herd certification standards to increase biosecurity in farms that produce deer urine. This program is administrated by the Archery Trade Association (ATA) and to date, no facility adhering to the ATA program standards has been found to have a CWD positive deer. To further ensure that deer urine products are safe CWD EvolutionTM LLC with help from Tink’s® and Wildlife Research® developed a next generation RT-QuIC test for natural deer urine products. The test mirrors the same testing protocol used to discover that CWD prions can be detected in deer urine and is more sensitive than currently deployed tests used to detect CWD.
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In 2019 Louisiana became the first state to ban deer urine products except for those that have been tested with the newly developed RT-QuIC test. In 2019 >50% of the deer urine sold in the US was tested by CWD EvolutionTM with the new RT-QuIC test. In 2020 many of the top manufacturers have committed to test urine used in their products. Products that have been RT-QuIC tested will be marked with the RT-QuIC tested checkmark logo. The combination of increased biosecurity in the ATA deer protection program and RT-QuIC testing demonstrates the strong commitment being made by manufacturers in the deer urine industry and is a compelling argument for the continued sale and safety of natural deer urine products. The partnership between the deer urine industry and CWD EvolutionTM is a success story of how CWD testing can overcome obstacles faced by deer farmers or manufacturers. LiVE AniMAL rT-QuiC TESTing: Both CWD EvolutionTM and the USDA are developing RT-QuIC testing as a useful tool for CWD detection in live animals as well as in post-mortem CWD testing. The challenge for live animal testing is that the samples that can be easily acquired, such as the lymphoid tissue of the tonsil or rectum, do not have the same levels of CWD as samples that can be taken post-mortem. In order to use samples with lower levels of CWD for detection we need a more sensitive test than is currently available. So far, there has been promising results using RT-QuIC for detection of CWD in live animals due to the vastly increased sensitivity achieved with RT-QuIC. The live animal RT-QuIC test results are still less sensitive than post-mortem testing but we are getting closer to a positivity rate that is useful for diagnostic purposes. There are a few challenges for this test to be brought to the USDA for approval. The RT-QuIC test uses a substrate that is sensitive to conversion by CWD prions. The production, quality control and validation of a substrate that can be shipped to labs for testing is one hurdle that CWD EvolutionTM is focused on solving. Another key metric that would demonstrate that RT-QuIC testing is useful and robust is testing a curated sample set across multiple laboratories with the same substrate kit and testing protocol to analyze reproducibility. CWD EvolutionTM is working with the USDA to develop a sample set that can be blinded and shared across laboratories and tested in 2020to determine the reproducibility of RT-QuIC testing.
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The RT-QuIC assay has the potential to enable testing that is not possible with the currently available testing methods for CWD. The increased sensitivity has the potential to test many types of samples such as degraded samples or improperly stored samples which can lead to more robust CWD monitoring. HuMiC ACiD AnD CWD: Chronic wasting disease is a grim problem with little good news to be shared. There are many problems that CWD poses for the deer farming industry. One challenge is when CWD has been found in a pen used to house deer, how long does it stay infectious or dangerous to deer being raised in the same space and what measures can be used to degrade CWD prions found in the soil or on surfaces in that pen. Like the disease itself the answer to this question is diďŹ&#x192;cult. There are many factors that influence the persistence of CWD in the environment. The first consideration is how much CWD is in the pen. Was there a single deer that was found to be lymph node only positive? In this case there may not be enough CWD deposited into the environment to pose a risk to deer in that space in the future. Were there CWD positive deer in the pen for years? Was the prevalence high (20-50% CWD(+))? Did deer die in the pen of CWD? Each of these questions and many more influence the potential persistence of CWD. We know that brain material can remain infectious for a very long time. We also suspect that prions once accumulated to a level that is infectious can remain that way for years if not a decade or longer. The challenge is that scientifically controlled experiments on that time scale are both very expensive and not commonly undertaken in the scientific community due to the length of funding available from granting agencies. There are a few scientific studies that have attempted to address the persistence of CWD in soil or on surfaces. A recent study by Kuznetsova et al. garnered a lot of attention by finding that an humic acid, an organic compound found naturally in soil, can not only degrade CWD prions in vitro but that infectivity was delayed after treatment of CWD(+) brain homogenates with humic acid. Other groups have had similar lines of investigation and had similar conclusions. It seems likely that soil components such as humic acid are capable of degrading CWD in the environment. In my CONTINUED ON NEXT PAGEÂť North American Deer Farmer
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laboratory I performed a series of RT-QuIC experiments in collaboration with the Michigan Deer Farmer Association that showed detection of CWD was decreased by treatment with humic acid. Many more experiments are needed but the initial finding was that detection is reduced in both prions found in soil naturally and diluted lymph node homogenate. Importantly, these finding only demonstrate that detection was eliminated not infectivity. In a recently published study, my colleagues and I demonstrated that repeated freezing and thawing of fecal pellets also reduced detection. It appears that CWD prions at lower concentrations are affected by many factors found in soil and experienced on the landscape. What is not known and remains a key question is can humic acid be applied to a contaminated pen and degrade CWD present to a level that would no longer pose a risk for deer placed in that pen. The answer likely is dependent on a lot of the factors mentioned above such as the levels of CWD present and how CWD is distributed in the pen. Humic acid may degrade all but a single hot spot and the result would end up the same as if no humic acid was used. There needs to be a series of controlled experiments where deer are placed in two sides of the same pen separated by a double fence and one side is treated with humic acid and the other is not. Even in this experiment we can not be sure that CWD was evenly found on both sides of the pen. Again, there are many factors that influence the outcomes of these long-term experiments some can be controlled and others remain difficult to account for. There has also been interest in determining if feeding humic acid to deer can prevent or delay prion disease. I am not aware of any study that clearly demonstrates that humic acid has any affect on ingested prions. I have heard anecdotal evidence that feeding of humic acid increases the body condition of deer but there is no scientific controlled study to support those claims. It may absolutely be a real affect but in the absence of controlled studies it remains an open question in my mind. We know very little about how the underlying health and body condition of deer affects the CWD disease course. It does seem possible and even likely that healthier deer may exhibit a longer CWD disease course but without controlled experiments we cannot be certain about the answer to that question. I do not think that feeding of humic acid can prevent eventual death from CWD or reverse CWD in an infected deer. I understand that what can 126
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be proven scientifically and what people may think can be very different but my focus in this article is to layout what has been concluded by scientists and which direction we can go to learn more. SEMEn AnD CWD The recent finding that CWD can be detected in the semen of positive bucks sent ripples though the deer farming community. A method called protein misfolding amplification or PMCA was used to detect CWD in semen. PMCA is a very sensitive prion amplifying technique which is similar to RT-QuIC. Both testing methods are capable of detecting CWD past a point where the sample would be infectious to both deer and when inoculated into the brain of a mouse. Currently, CWD has only been detected in semen and not shown to be infectious. However, state agencies have reacted strongly to natural deer urine products in spite of the fact that no natural infection has even been demonstrated via urine. There is an ongoing experiment where mice have been inoculated with positive semen to determine if infectious prions are present. I would not be surprised if that study did in fact find infectious levels of prions in semen. The next step, which is unlikely to be resolved soon, is to determine if a CWD infection can be established in deer by introduction of semen in a natural way. The undertaking of such an experiment would be expensive and therefore remains unlikely. To date, there has not been a case where semen was identified as the source of a CWD outbreak. Working with NADeFA, Dr. Haley and I have proposed a study to determine if we can develop a RT-QuIC based test to ensure the safety of semen. We feel that it is important to stay ahead of this issue and have an option in place that may assuage the concerns of governing agencies to allow continued free transport of semen. u nADeFA.org
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» CWD RESEARCH
Humic Acid degrades CWD prions in Soil! By Dan Harrington
CWD is a disease that seems to be increasing in prevalence throughout North America and beyond. This is especially important to deer farmers who are affected by burdensome regulations and restrictions. It can ultimately put us out of business if we have the misfortune of having a positive test. It has definitely stifled our once overwhelming enthusiasm for what we do. There is hope. Although the route of transmission is still unknown, it has been hypothesized that CWD can be transmitted indirectly through environmental contamination, most commonly believed, through contaminated soil. The CWD prion is thought to be very resilient. It has been shown to exist in soils for many years. The Centers for disease control (CDC) states that the CWD prion can remain in the soil for a very long time. It is termed PrP resistant, because of its resistance to harsh chemical treatments, as well as its inability to be degraded naturally in 130
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the body. Autoclaving, a common method of sterilization has proven ineffective on the CWD prion. Along comes humic Acid. Humic acid is a naturally occurring component of soil. Through years of abusing our soils with herbicides, pesticides and pollution we have depleted the once sufficient amounts in our soils. In 2018, Judd Aiken at the University of Alberta demonstrated that humic acid at various concentrations degraded the CWD prion in infected brain tissues. Even though, humic acid degraded the prion in brain tissues, Aiken was un-able to conclude that humic acid would degrade the prion in soil, due to the complexity of soils. Many of us believed that humic acid would degrade the prion in the soil and wanted to test this theory. If confirmed we believed it would give deer farmers a major tool to fight CWD. There were many skeptics, but the United Deer Farmers of Michigan was not one of them. They stepped up nADeFA.org
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and committed to funding a project, to verify if humic acid only find prions in 12% of the samples, using state of the art was capable of degrading prions in the soil. technology. Keep in mind that the samples were taken in high congregation areas around feeders and waterers. METHoDS Wilderness Whitetails of Wisconsin, a longtime leader Recent studies with Scrapie, a prion disease of sheep, has in the Deer Farming community agreed to allow testing at one of their facilities. Greg and Shorty Flees had already shown that differing soil types can present either inhibitory been involved with helping to find a solution to CWD, results or no difference in the replication efficiencies through their involvement in resistance research with Dr. depending on soil composition (Saunders it al. 2011). High Haley. Testing sites were focused on feeding and watering organic matter soils showed greater reduction in detections areas. Samples were taken with a 1 inch diameter stainless where high sand soils showed no apparent reduction in steel soil sampling probe at depth of 1 inch. Twenty Four replication efficiency (Saunders et al. 2011). This may be samples were taken. These samples were packaged and sent explained in this study, in that High organic matter soils have to CWD Evolution for prion testing. higher humic acid content vs sandy soils, which have very little humic acid. rESuLTS Testing was done using RT-QuIC. The initial testing indicated that 3 of the 24 samples contained prions. The samples were retested with the same results. These positive samples were then treated with humic acid. The humic acid was dissolved in deionized water at the equivalent concentrations of those used in the Aiken study for comparison purposes. These concentrations were the equivalent values of 2.5 grams/ liter, 15 grams/liter and 25 grams per liter of humic acid. The humic acid source did vary from the Aiken study. Obtaining pure chemical grade humic acid was expensive and problematic. It would also be cost prohibitive for future practical application. The decision was made to use the New Mexico Mesa Verde Humates source, seeing this is easily available and affordably priced. The incubation period was also extended from 24 hours to 72 hours, reasoning that this increased period is not significant to practical deer farming purposes. All samples treated tested negative for CWD prions. The humic acid at the highest concentration (25 g/l) interfered with positive results and therefore the negative values obtained at that concentration could not be used. However, all samples treated tested negative so we could assume that the highest concentration would also be negative at 25 g/l. These results show a 10 fold increase in the degradation of the prion at the extended incubation period used, verses the Aiken study.
going ForWArD I believe this study showed that if Deer Farmers want a solution to CWD, we are going to have to figure it out on our own. Currently, with the help of Scott Follett, we are setting up a study in Wisconsin at Apple Creek Whitetails. This study will look at the life expectancies of CWD positive deer on humic acid feed. It will test prion shedding on CWD positive deer fed Humic acid. It will test prion presence in semen of CWD positive deer fed humic acid. It will also continue exploring mineral levels and their effect on CWD. Finally it will also test the effects of CWD on markered deer. If you or your organization would like to participate in helping to solving CWD, please go to the website at www.solvecwd.com. You can also call Dennis Simpson at 313-218-0105. Whitetails of Wisconsin has already committed to supporting this project.
ConCLuSionS First, I’d like to thank the Deer farmers of Michigan and Wisconsin for their forward thinking, and generosity in funding these studies. Finally, we’ve already covered this in other articles, but if you haven’t, think about using humic acid in your operation, and do your part to prevent CWD. Feel free to contact me at any time if you’d like to discuss inTErESTing THougHTS Prions may not be as hardy as previously thought. The anything. My cell is 906-282-7555, or email me at facility tested has a 70% CWD prevalence rate and we could dan@wideandhighwhitetails.com. u Spring 2020
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ADVERTISER’S INDEX Addison Biological Laboratory, Inc. .........................62 ADM Alliance Nutrition ...........................................67 Ani-Logics Outdoors ..............................................128 Arrow Ridge Ranch LLC ...........................................74 Blackjack Whitetails .................................................33 Blue Creek Whitetails ...............................................17 Brazos Valley .......................................................19, 99 C&E Wildlife Products .............................................65 Cedar Breaks ............................................................55 Cervid Livestock Foundation....................................66 Cervid Solutions .......................................................66 Classic Canyon Ranch...............................................41 Cross Canyon ............................................................11 Dan Inject Dart Guns ................................................34 Deer Breeder Corp....................................................80 Deer Tracking Magazine .........................................135 Deer and Wildlife Stories............................................9 Double H Whitetails ...........................................46,47 Deerstore ..................................................................88 Eaton Highland Farm ................................................45 Enable USA ...............................................................18 EZID.........................................................................60 GMS .........................................................................21 Headgear...................................................................79 Hidden Antler R anch ..............................................133 High Roller Whitetails ..............................................63 Keeper R anch .............................................................3 Mayes Elk Farm.......................................................129 MaxRax.....................................................................88 Medgene ...................................................................20
MVP Whitetails ..............................................136, IBC NADR ..................................................................52,53 Nature Calls Inc ..........................................................5 Nature’s Formula ......................................................75 Nutra-Glo ...............................................................134 Oregon Ag ................................................................87 PetAg ........................................................................77 Pneu-Dart ..........................................................IFC, 1 Prime Management Acres .........................................26 Purina Mills ..............................................................15 Record Rack - Cargill ...............................................73 Rocky Ridge Whitetails ............................................81 RW Trophy................................................................89 SCI ...........................................................................54 Schafer Whitetails...................................................122 Shock Effect..............................................................97 Southeast Trophy Deer Association ..........................61 Springfield Whitetails.............................................123 S&S Whitetail Galore..............................................113 Storm Ends Whitetails ............................................114 Tajada Whitetails .....................................................BC Thunderbay Whitetails ...........................................103 Top Gun Whitetails.....................................................4 Triple C Trophy Whitetails .................................8,132 Vara Ranch ..........................................................39, 96 Way to Go Whitetails ................................................35 White Ghost Ranch.................................................127 Whitetail Syndication .............................................115 Wildlife Pharm/Zoo Pharm ......................................40
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North American Deer Farmer
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nADeFA.org
Spring 2020
Spring 2020
nADeFA.org
North American Deer Farmer
135
North American Deer Farmer
136
