COME in PAIRS VETERINARY Veterinary

E Q SOMETIMES MIRACLES... UINE equine American Edition | February 2020 COME IN PAIRS VETERINARY veterinary

EDUCATION/American education Edition

Volume 32 Number 2

AND

TOGETHER, ASSURE GUARD GOLD-NG AND ASSURE GUARD GOLD

CREATE A POWERHOUSE AGAINST YOUR MOST CHALLENGING DIGESTIVE CASES. in this issue: February

USE ASSURE GUARD GOLD-NG FOR FAST RELIEF AND MAINTAIN EXCELLENT DIGESTIVE Beyond good intentions: The ethics of ‘spotting’ medications to colleagues The official journal of the HEALTH WITH ASSURE GUARD GOLD. American Association of Theiler’s disease associated with administration of tetanus antitoxin contaminated 2020 Equine Practitioners, produced with nonprimate (equine) hepacivirus and equine parvovirus-hepatitis virus Ask your Arenus Veterinary Solution Specialist how Assure Guard Gold-NG in partnership with BEVA. and Assure Guard Gold can help your equine patients quickly and Treatment of haemoperitoneum secondary to ruptured granulosa cell effectivley recover from the digestive upsets you treat daily. tumours in two mares

Arenus Animal Health | 866-791-3344 | www.arenus.com equine veterinary education American Edition FEBRUARY 2020 • Volume 32 • N umbER 2

AAEP NEWS In this issue contents Beyond good intentions: The ethics of ‘spotting’ medications to colleagues.. III

Glanders Guidelines released on AAEP website, publications app...... V

Resolving conflict in a healthy way...... VIII

Highlights of Recent Clinically Relevant Papers S. WRIGHT...... 58

Editorial Equine welfare A. ATOCK...... 60

Case Reports Theiler’s disease associated with administration of tetanus antitoxin contaminated with nonprimate (equine) hepacivirus and equine parvovirus-hepatitis virus J. J. KOPPER, H. C. SCHOTT II, T. J. DIVERS, T. MULLANEY, L. HUANG, E. NOLAND and R. SMEDLEY...... 62

Locally invasive melanoma in the internal laminae of the hoof of a bay mule G. KAY, P. MCNEIL, D. KNOTTENBELT and N. TLIGUI...... 66

Treatment of haemoperitoneum secondary to ruptured granulosa cell tumours in two mares F. C. F. WORSMAN, S. Z. BARAKZAI, M. P. DE BONT, S. TURNER and L. M. RUBIO-MARTÍNEZ...... 71

Septic pyomyositis, multiple pelvic osteomyelitis and thrombosis in a Thoroughbred foal M. C. PRESSANTO, F. BECCATI, V. STEFANETTI, F. PASSAMONTI, N. PILATI and M. PEPE...... 78

Imaging findings associated with nasopharyngeal amyloidosis in an Arabian stallion L. A. RUSSELL, C. NAVAS DE SOLIS, C. M. WHITFIELD-CARGILE, M. A. DEVEAU, B. R. BUCHANAN and L. J. GILMOUR...... 79

Clinical Commentaries Theiler’s disease T. J. DIVERS and J. E. TOMLINSON...... 63

Equine external beam radiation therapy: Techniques and challenges N. C. DESAI and E. M. GREEN...... 84

Original Articles Atypical myopathy in the South-East of England: Clinicopathological data and outcome in hospitalised horses B. DUNKEL, A. RYAN, E. HAGGETT and E. J. KNOWLES...... 90

Evaluation of a pinhole castration technique in ponies: Comparing single with double ligation (using silk or catgut) of the spermatic cord M. Y. MIR, M. R. FAZILI, K. H. DAR, M. S. MIR and S. QURESHI...... 96

Review Article How to critically appraise a paper G. L. PINCHBECK and D. C. ARCHER...... 104

Critically Appraised Topic Can ocular administration of atropine cause colic? R. COOMER...... 110

Advertisers’ Index...... 112 Cover photo by Dr. Pamela Corey. American Association of Equine veterinary education Equine Practitioners American Edition 4033 Iron Works Parkway Lexington, KY 40511 FEBRUARY 2020 • Volume 32 • Number 2 TEL (800) 443-0177 • (859) 233-0147 FAX (859) 233-1968 EMAIL [email protected] Editor (UK) Assistant Editors aaep.org T. S. Mair, BVSc, PhD, DEIM, DESTS, F. Andrews S. Love D. Archer M.L. Macpherson To access our website, go to aaep.org, select DipECEIM, MRCVS LOGIN, then enter your email and password. If you F.T. Bain M.J. Martinelli have difficulty logging in or have forgotten your Editors (USA) A.R.S. Barr I.G. Mayhew password, please call or email the office. N. A. White II, DVM A. Blikslager M. Mazan W. D. Wilson, MRCVS M. Bowen C.W. McIlwraith AAEP Officers N. Cohen B. McKenzie David Frisbie, DVM, President Deputy Editors V. Coudry R. Moore Y. Elce A. Dart M. Oosterlinck Scott Hay, DVM, President-Elect P.R. Morresey J.-M. Denoix A. Parks Emma Read, DVM Vice President P.A. Wilkins T. Divers S. Puchalski Lisa Metcalf, DVM, Treasurer P. Dixon C. Riggs Jeff Berk, VMD, Immediate Past President Management Group W. Duckett H. Schott D. Foley B. Dunkel J. Schumacher AAEP Staff T. S. Mair S. Dyson S. Semevelos David Foley, CAE, Executive Director N. A. White T. Fischer J. Slater D. Freeman B. Sponseller [email protected] W. D. Wilson T. Greet C. Sweeney Lori Rawls, Director of Finance & Operations J. L. N. Wood [email protected] R. Hanson H. Tremaine P. Harris K. Wareham Sally J. Baker, APR, Director of Marketing Management Board & Public Relations • [email protected] M. Hillyer S. Weese A. R. S. Barr C. Scoggin Keith Kleine, Director of Industry Relations M. Holmes R. Weller [email protected] D. Foley N. A. White (US Editor) N. Hudson C. Yao D. Mountford S. White Nick Altwies, Director of Membership P. Johnson [email protected] T. S. Mair (Editor) W. D. Wilson (US Editor) P.T. Khambatta Ex-officio S. E. Palmer J. L. N. Wood (Chairman) J.-P. Lavoie J. Cooney Kevin Hinchman, Director of Information Technology [email protected]

Karen Pautz, Director of Education Equine Veterinary Education is a refereed educational journal designed to keep the practicing veterinarian up to [email protected] date with developments in equine medicine and surgery. Submitted case reports are accompanied by invited reviews Sadie Boschert, Student Programs Coordinator of the subject (satellite articles) and clinical quizzes. Tutorial articles, both invited and submitted, provide in-depth [email protected] coverage of issues in equine practice.

John Cooney, Publications Coordinator Equine Veterinary Education (American Edition ISSN 1525-8769) is published monthly by the American [email protected] Association of Equine Practitioners, an international membership organization of equine veterinarians. Office of Giulia Garcia, Communications Coordinator publication is 4033 Iron Works Parkway, Lexington, KY 40511. Periodicals Postage paid at Lexington, KY and [email protected] additional mailing office. POSTMASTER: Send address changes to: Equine Veterinary Education, 4033 Iron Works Parkway, Lexington, KY 40511. Megan Gray, Member Concierge [email protected] Communications regarding editorial matters should be addressed to: The Editor, Equine Veterinary Education, Dana Kirkland, Sponsorship & Advertising Mulberry House, 31 Market Street, Fordham, Ely, Cambridgeshire CB7 5LQ, UK. Telephone: 44 (0) 1638 720250, Coordinator • [email protected] Fax: 44 (0) 1638 721868, Email: [email protected]. Katie McDaniel, EDCC Communication Manager All manuscript submissions for the journal should be submitted online at http://mc.manuscriptcentral.com/eve. Full [email protected] instructions and support are available on the site and a user ID and password can be obtained on the first visit. If you Deborah Miles, CMP, Trade Show Coordinator require assistance, click the Get Help Now link that appears at the top right of every ScholarOne Manuscripts page. [email protected] All subscription inquiries should be addressed to: Subscriptions Department, AAEP, 4033 Iron Works Parkway, Jayson Page, Office Manager Lexington, KY 40511, Telephone: (859) 233-0147, Email: [email protected]. Subscription rates: AAEP annual [email protected] membership dues include $40 for a subscription to Equine Veterinary Education. Other subscriptions at $151.80. Single copies $37.50. Paul Ransdell, Senior Development Officer [email protected] Canadian Subscriptions: Canada Post Corporation Number 40965005. Send change address information and Carey Ross, Scientific Publications Coordinator blocks of undeliverable copies to IBC, 7485 Bath Road, Mississauga, ON L4T 4C1, Canada. [email protected] © World copyright by Equine Veterinary Journal Ltd 2020. Pam Shook, Foundation Programs Coordinator [email protected] The authors, editors and publishers do not accept responsibility for any loss or damage arising from actions or Sue Stivers, Executive Assistant decisions based or relying on information contained in this publication. Responsibility for the treatment of horses [email protected] under medical or surgical care and interpretation of published material lies with the veterinarian. This is an academic publication and should not be used or interpreted as a source of practical advice or instruction. Amity Wahl, Communications & Technology Coordinator The American Association of Equine Practitioners cannot accept responsibility for the quality of products or ser- [email protected] vices advertised in this journal or any claim made in relation thereto. Every reasonable precaution is taken before Kristin Walker, Membership & Event advertisements are accepted, but such acceptance does not imply any form of recommendation or approval. Services Coordinator [email protected] All companies wishing to advertise in Equine Veterinary Education, American edition, must be current AAEP exhibitors. AAEP retains the right, in its sole discretion, to determine the circumstances under which an exhibitor may advertise in this Elaine Young, Convention & Meetings Coordinator journal. While all advertisers must comply with applicable legal guidelines, Compounding Pharmacies are specifically [email protected] directed to limit themselves to pharmacy practices as dictated by the FDA Center for Veterinarian Medicine, Compliance Policy Guideline (www.fda.gov/ora/compliance_ref/cpg/cpgvet/cpg608-400.html). Advertising any complete or partial Published monthly. Deadlines are the mimicry of drugs and dosage forms of FDA approved formulations will not be accepted. Compounding Pharmacies, or any seventh of the preceding month. other exhibitors/advertisers who violate this rule in any fashion, will render their advertising contract null and void. Address advertising inquiries to Dana Kirkland (859) 233-0147 / [email protected] As a private organization, the AAEP reserves the right to exclude any company from advertising in Equine Veterinary Education, American edition, for any reason. The signing and delivery of the advertising contract shall AAEP Mission Statement: To improve the health and constitute an offer subject to acceptance by the AAEP. In its sole and absolute discretion, the AAEP may revoke its welfare of the horse, to further the professional acceptance of the advertising contract or may terminate any contract by delivery of written notice, in which event development of its members, and to provide resources the AAEP shall have no liability to the advertiser for damages for any other remedy. and leadership for the benefit of the equine industry. Printed by: Cenveo Publisher Services, Lancaster Division, Lancaster, PA. ETHICS AAEP News February 2020 III

Beyond good intentions: The ethics of ‘spotting’ medications to colleagues By Nick Huggons, DVM, DACVS

In-house pharmacies in a provisions of the act. Reselling the plasma, however, referral center are generally would place the clinic as a functioning blood bank in stocked with a more diverse California with all of the attendant regulations. The range of prescription medica- exemptions are not broad enough to allow reselling of tions due to the center’s need biologics under the current law without obtaining proper to triage and treat a broad licensure. This would require a license pursuant to the and varied caseload. CFAC Commercial Blood Banks for Animals and Biologics chapter 1.5. Further, the clinic would then be Frequently, a referral center is liable for potential negative reactions or diseases that contacted by an ambulatory could be transferred. veterinarian regarding a case that may not result in physical If the drug is not a biologic and considered dangerous Dr. Nick Huggons referral of the patient, yet (“federal law prohibits dispensing without prescription”), requires treatment that there is nothing in the California Veterinary Medicine includes prescription medications not typically stocked by Practice Act to prohibit such activity. It is recognized as the ambulatory veterinarian. common practice to “spot” a colleague for drugs they require. However, the AVMA’s Principles of Veterinary For example, an ambulatory veterinarian with a limited Medical Ethics require a VCPR before a veterinarian can number of reproductive cases attends a foal for their write a prescription for an animal patient. In addition, client and determines that it has failure of passive each state’s veterinary medical board further regulates transfer. The client is not willing to refer at this time and how prescriptions must be written. This, in turn, dictates the veterinarian does not have a supply of plasma and that the rules will vary by state—several states allow vet- fluids to administer the foal; however, they need to erinarians to fill prescriptions from other veterinarians in administer these in a timely manner. A simple call into the certain scenarios while others do not. local referral center locates a supply of frozen plasma and fluids that can be furnished. Being a newborn, the foal As a veterinarian dispensing drugs with a valid VCPR, the does not have patient status with the referral center, even clinic is not regulated by the California Board of if the center is familiar with the mare. There is no Pharmacy if it is not filling prescriptions for other veteri- Veterinarian-Client-Patient Relationship (VCPR) estab- narians’ patients. Controlled drug transfers from one lished between the referral center and the foal. However, DEA-registered veterinarian to another DEA-registered as has been common practice, the referral center discusses veterinarians are permitted. This mandates records of the the case with the referring veterinarian and dispenses the transfers by log entries and invoices for CIII-V drugs; in much-needed plasma and intravenous fluids. addition, CII drugs must be accompanied by DEA Form 222 issued by the recipient and completed by you with a What are the implications behind a local referral center copy to the DEA’s local field office. The issue of reselling in-house pharmacy dispensing a biologic (“non-danger- drugs becomes more complicated if significant quantities ous” or “non-prescription” drug) versus a controlled are being resold to other resellers; practitioners should (“prescription” or “dangerous” drug) under these or avoid these scenarios. (All of this, however, is different similar circumstances? than one DEA-registered veterinarian directly dispensing medications to another DEA-registered veterinarian’s The answer depends on the product and each state’s patient without the dispensing veterinarian maintaining a veterinary practice rules. VCPR with said patient.)

Plasma is not a dangerous drug; it is a biological that, in Check your own state’s veterinary practice rules to avoid my state of California, is regulated by the California Food confusion on this issue. Biologics versus prescription and Agriculture Department. Section 9272(3) of the drugs are likely to be covered under different licensing California Food and Agricultural Code (CFAC) exempts bodies within each state, and veterinarians must be aware licensed private veterinarians who collect blood or blood of the nuances between biologic and prescription products solely for use in their own practices from products that may be called upon by fellow practitioners.

Dr. Huggons is co-owner of San Luis Rey Equine Hospital in Bonsall, Calif., and a member of the AAEP’s Professional Conduct and Ethics Committee. IV AAEP News February 2020 Association

5 things to know about AAEP this month

1. Read the AAEP’s new 2. Promote the value of your 3. Enjoy free access through 4. Access digital versions 5. Join the more than 1,000 Glanders Guidelines on the practice’s dental services early March to the EVE of EVE, Proceedings, members discussing AAEP Publications App or to clients with AAEP’s and EVJ articles from guidelines and more using veterinary and industry topics online at aaep.org/sites/ ready-to-use educational Kester News Hour and the the free AAEP Publications in the AAEP Member Vet Talk default/files/Documents/ resources accessible at Milne Lecture at App. Search “AAEP Facebook group. Search for AAEPGlandersGuidelines.pdf. aaep.org/dentalservices. https://tinyurl.com/kest19. Publications” at your the group and click “Join.” app store.

Reward excellence with an AAEP award nomination Deadline to nominate is June 1

Honor a colleague who is serving the profession, association or welfare of the horse in outstanding ways with a 2020 AAEP annual award nomination. The AAEP is accepting nominations in the following categories until June 1:

The Distinguished Educator – Academic Award honors an individual educator who by his or her actions and commitment has demonstrated a significant impact on the development and training of equine practitioners.

The Distinguished Educator – Mentor Award honors an individual who by his or her actions and commitment has demonstrated a significant impact on the development Dr. David Wilson, left, accepts the Distinguished Educator – and training of equine practitioners through mentoring. Academic Award from 2019 AAEP President Dr. Jeff Berk during the 65th Annual Convention in Denver, Colo. The Distinguished Life Member Award recognizes a member who has demonstrated outstanding or extraordi- onstrated exceptional compassion or developed and nary service to the AAEP over the course of their career. enforced rules and guidelines for the welfare of horses.

The Distinguished Service Award recognizes an individual The AAEP Research Award recognizes an individual who who has provided exemplary service to the AAEP or a has recently completed research that has or will make a similar organization to the benefit of the horse, horse significant impact on the diagnosis, treatment or industry or the profession of equine veterinary medicine. prevention of equine disease. Nominations are open to all individuals whose research is acknowledged by presenta- The George Stubbs Award recognizes the contributions tion or publication and by peer review as a significant made to equine veterinary medicine by individuals other advancement in equine medicine or innovation in equine than veterinarians. science. Nominees must have had their research presented or published during the two years prior to when nomina- The Sage Kester Beyond the Call Award is named in tions are submitted to the AAEP. honor of its first recipient, the late General Wayne O. “Sage” Kester, DVM, and represents the highest honor Visit aaep.org/about-aaep/annual-awards for nomination bestowed by the AAEP upon a current or former member. forms as well as additional information about the awards The award is presented to an individual who has made and selection process. Nomination forms are also significant and long-lasting contributions to equine available from Sue Stivers at (859) 233-0147 or sstivers@ veterinary medicine and the community. aaep.org.

The Lavin Cup (The Equine Welfare Award) recognizes a Award recipients will be honored at the AAEP’s 66th non-veterinary organization or individual that has dem- Annual Convention in Las Vegas, Nev., Dec. 5–9, 2020. Association AAEP News February 2020 V

Member search? There’s an app for that

Do you need to contact an AAEP colleague but are not sure of their email address or phone number? There’s a simple solution—the AAEP Publications App.

Like its name suggests, the AAEP Publications App is your digital source for the association’s catalog of publications, including Equine Veterinary Education, Annual Convention Proceedings, Foundation newsletter, annual report, white papers, guidelines and more. In addition, a “Member Lookup” button within the app enables you to conduct a quick and simple search by name of the membership database.

What previously required thumbing through 250+ pages of member listings in the discontinued print membership directory can now be accomplished in seconds from anywhere with Internet access using the app.

Download the app at no charge by searching “AAEP Publications” at the App Store or Google Play.

Glanders Guidelines released on AAEP website, publications app

The AAEP has published comprehensive guidelines for the “Although Glanders was officially eradicated in the United identification, control and prevention of Glanders, a States in 1934, it continues to be present in many contagious disease largely eradicated from the developed countries throughout the world,” said Dr. Sage, Richmond world but which has resurfaced recently with reported staff veterinarian for the Virginia Department of cases from countries in Asia, Africa, the Middle East and Agriculture and Consumer Services. “We must remain South America. The guidelines are accessible on both the vigilant through the USDA’s strict testing of horses at AAEP website and publications app. points of entry into the U.S. to prevent re-introduction of this disease.” Although long eradicated in North America, Australia and Europe, recent outbreaks in countries in which Glanders Prompt identification, euthanasia and appropriate disposal was previously eradicated indicate that the disease is no of all positive cases of the disease is critical to effective longer as geographically restricted in terms of its global prevention and control of Glanders. Although antibiotic distribution. This re-emergence, according to guidelines treatment of affected animals is employed in some areas of co-author Dr. Abby Sage, underscores the importance of the world where Glanders is endemic, it is not the accepted effective import control measures. option for most countries when dealing with an outbreak.

The Glanders Guidelines were co-authored for the AAEP’s Infectious Disease Committee by Drs. Sage and Peter Timoney, the Frederick Van Lennep Chair in Equine Veterinary Science at the University of Kentucky’s Gluck Equine Research Center. UAE View the Glanders Guidelines or save them as a PDF to

Dubai, your phone or tablet for future reference at aaep.org/sites/ default/files/Documents/AAEPGlandersGuidelines.pdf. In CVRL, addition to Glanders, AAEP guidelines for 19 other equine infectious diseases are available at aaep.org/guidelines/

Wernery, infectious-disease-control/using-guidelines. All AAEP

U. guidelines are also accessible on the free AAEP Publications App. Search “AAEP Publications” at your app store to download. Courtesy An extensive eruptive lesion on the cheek of a Glanders- infected horse. VI AAEP News February 2020 Association

Deadline nears to submit papers for 2020 convention in Vegas Papers due March 16 at 3:00 p.m. ET

Eligible for consideration are scientific papers, “how-to” 66th Annual Convention papers, review papers, abstracts ≤ 250 words and The Business of Practice papers. All paper presentations are VEGAS limited to 15 minutes with an additional 5 minutes for December 5-9, 2020 Q&A. Mandalay Bay Resort Papers must be submitted by March 16, 3:00 p.m. ET, at https://s3.goeshow.com/aaep/annual/2020/AAEP_paper_ Time is running out to submit a paper to be considered submission.cfm. Authors should visit the site in advance for presentation during the 66th Annual Convention in to set up a profile and provide paper and author informa- Las Vegas, Nev., Dec. 5–9. The presenting author of tion before uploading the paper when it is finished. selected papers will receive complimentary registration Complete considerations and ethical guidelines are and a stipend to support travel to the meeting. available in the General Instructions area of the site.

On air: EVE and EVJ podcasts

New episodes of the Equine Veterinary The EVJ podcast episode discusses the paper, Education and Equine Veterinary Journal “Radiographic and arthroscopic features of third carpal podcasts offer deeper looks into recently bone slab fractures and their impact on racing perfor- published papers. mance following arthroscopic repair in a population of racing Thoroughbreds in the UK.” An abstract of the paper In the EVE podcast episode, Dr. Solenn Le Corre discusses is accessible at ncbi.nlm.nih.gov/pubmed/31356679. the paper “Cor pulmonale secondary to pulmonary fibrosis in a 16-year-old mare.” The paper is accessible at The bottom line: Fracture configurations can be identified https://beva.onlinelibrary.wiley.com/doi/full/10.1111/ radiographically but is not a reliable predictor of commi- eve.12986. nution or other intra-articular lesions. Arthroscopy not only directs repair but also identifies and facilitates The bottom line: Cor pulmonale is rarely observed in management of concurrent lesions. The results reported horses, and there is very little published information about should assist in formulating appropriate prognoses for prognosis and outcome of this cardiac remodelling over Thoroughbred horses racing in the UK. the long term. The treatment of the underlying cause is essential to prevent any worsening of the lesions that could Listen to the 7-minute episode and subscribe to future lead to a global cardiac insufficiency. episodes at evj.podbean.com.

Download or listen to the 13-minute episode at equineveterinaryeducation.podbean.com.

MEMBERSHIP

Welcome new members, and congratulations recent graduates

New Members: Recent Graduates: John Canty, DVM, Middleburg, FL Emily Hughes, DVM, North Richland Hills, TX Leland T. Thomas, DVM, Saint John, NB, Canada Katie Gottleib, DVM, Newtown, CT Emily Wilson, DVM, Vacaville, CA Sierra Chanutin, DVM, Lake Worth, FL Stephanie Ann Butler, DVM, Lexington, KY Ashley Elaine Steuer, DVM, Lexington, KY MEMBERSHIP AAEP News February 2020 VII

Benefit: Find the perfect position or candidate in the AAEP Career Center

Whether you’re an associate seeking a new opportunity or a practice owner seeking qualified candidates for an open position, the AAEP Career Center will help you make a career connection.

The Career Center provides a targeted and cost-effective means for employers to reach qualified candidates, with rates significantly below the mass market job boards. For just a little more, employers can expand the reach of their job post through inclusion in the Veterinary Career Network, which will push their post out to nearly 60 veterinary-related sites like the AVMA, many state VMAs and other VCN participants.

After posting a job opening, employers can proactively search resumes on file in the Career Center using multiple criteria to identify potential candidates. They will also receive email notice when job seekers apply for the posted position.

Job seekers, meanwhile, can view all available openings or narrow their search by location, keyword or other criteria; sign up for job alerts to receive notice when new positions of interest are posted; upload an anonymous resume available for review by employers; and take advantage of resources to assist with their job search such as assistance with effective resume and cover letter writing, salary negotiation and more.

Post your job opening or your resume in the AAEP’s Career Center today at jobs.aaep.org. If you have questions about the Career Center or need assistance crafting a job post, contact Megan Gray, AAEP’s member concierge, at [email protected].

Ophthalmology June 24-27, 2020 • Gainesville, Florida

For more information, visit aaep.org/meetings VIII AAEP News February 2020 WELLNESS

Resolving conflict in a healthy way By Kevin Foote, LMSW and Ted Fish, Ed.D., MBA

Everyone knows the story: Imagine a clinic where instead of joining together to take Two doctors who know each on the competition, doctors are taking out each other. other reasonably well start a Imagine a bunch of football players, who as soon as the company. They have similar whistle blows, tackle each other instead of the opposite financial goals. They are in team! Wouldn’t you love to take on a team like that for parallel places in their careers. market share? Unfortunately, this scenario is all too They are eager, ambitious, and common in equine practices. they work their tails off— putting up with long hours, When conflict escalates beyond what can be managed, a difficult clients and times third party is needed. The person has to come from the when they’re not sure how the outside. The antagonists are in a tug-of-war, and there’s Kevin Foote bills are going to be paid. too much pressure on the rope. Anything anybody says is a reason to pull in the opposite direction. What the profes- Somehow, they succeed. The books move from red to sional does is listen to each of the sides, and then once the black to solidly profitable. Their reputations in the rapport has been built, they demand that both stop community grow. Associates are hired—good doctors, who tugging the line. look to be interested in buying in. The professional interrupts And then the train comes off the tracks. the pattern of conflict and …if we can become helps each walk to the other’s invested in a shared The minor things, the little annoyances that they always position, to see how life managed to tolerate in each other no longer appear small. looks from where that person vision, it can give us Cracks threaten to become rifts. They can’t stand meeting stands. The purpose is not to reason to commit together. Maybe one micromanages everything the other persuade anyone of anything ourselves in a does. Maybe one fixes the schedule so she doesn’t have to but to listen and understand. be on call. Maybe one hoards the clients for himself or Many times, people just want relationship of criticizes the other behind her back. The more each thinks to be heard. Then, new professional respect. about it, the more they begin to wonder how the two of agreements can be crafted them can co-exist. What can they do? from common ground.

The details of conflict vary from practice to practice. In This is not a novel skill. All of us do this all the time—in some companies, owners are pitted against the staff. You our marriages and in relationships where we are invested. simply can’t get them to be thoughtful or accountable We have to remember that our professional relationships enough in their work. In other companies, the rift is with are a vehicle. They are not marriages. They are not until the associates. They say they work too hard, get paid too death do you part. But if we can become invested in a little, criticize everything you do. When conflict escalates, shared vision, it can give us reason to commit ourselves in communication breaks down and a vicious cycle builds. a relationship of professional respect.

Conflict is actually natural. People are different, and When I go to organizations full of conflict, I usually find expressing different needs and different points of view is that the people are getting a bad rap. Escalating tensions one of the signs of a healthy company. But when conflict often arise from systems failures. People need support. reaches the point where it is causing high blood pressure, Type A personalities—high achievers who are used to migraines, self-medication, or that feeling of dread being the best—need to know when something is their whenever you think of seeing that person at work; when it business and when it is not. Setting clear chains of reaches the point that it becomes an elephant in the room, command along with systems of accountability, like drawing energy away from the board, doctor or staff monthly meetings, financial reports, and appropriate meeting, then it has become dangerous. Unless it is dealt checks and balances, help capable people stay out of each with, the conflict will create harm for your organization other’s yards. and your health. continued on next page

Kevin Foote, LMSW, is President of Footeworks, a company in Mattituck, N.Y., that helps business owners and their staffs produce the results they want in their professional and personal lives. Ted Fish, Ed.D., MBA, formerly with Footeworks, is executive director of the Gardner Carney Leadership Institute in Colorado Springs, Colo. WELLNESS AAEP News February 2020 IX

Banfield releases suicide-prevention training as a free resource

Banfield Pet Hospital has made its suicide-prevention training, “ASK – Assess, Support, Know,” available as a free resource to the veterinary profession. The first of its kind, Banfield designed ASK specifically for veterinary professionals to help them recognize and address emotional distress and suicidal thoughts.

According to the Centers for Disease Control and Prevention (CDC, 2015) one in six veterinarians considers suicide. Further, the CDC found rates of suicide are 2.1 times higher than the general population among male veterinarians and 3.5 times higher for female veterinarians.

You can access ASK at ASKAssessSupportKnow.com. The training includes real-life scenarios, talking points, myth-busting and other resources to help guide veterinary professionals in navigating tough situations.

Resolving conflict in a healthy way, continued

Healthy organizations establish working channels of com- Keys to handle the conflict: munication in well-run meetings with fixed agendas. They • Bring in an objective third party to let the pressure carefully attend to their young and old, making equitable off the system. and transparent plans that guide buy-ins and buyouts, so • Instead of tugging in a war, reduce the tension by the next person in line feels safe and eager to play. joining their vantage point. Healthy organizations invest in culture. When conflict is • Craft a shared vision about where you are going. acute and hazardous, the pressure must be taken off. • Invest in the long-term health of your company Ultimately, however, the key to conflict resolution is a by establishing clear governance structures, checks system that supports long-range relationships and and balances, and meetings that encourage open long-term health. communication.

July 27-29, 2020 • Lexington, KY Featuring:

Podiatry Sport Horse Pre-Purchase Exam

For more information, visit aaep.org/meetings X AAEP News February 2020 FOUNDation

The Foundation for the Horse awards $366,000 in scholarships

Thirteen veterinary students and four veterinary school graduates of 2019 received early holiday gifts during the AAEP’s 65th Annual Convention in the form of scholarships from The Foundation for the Horse and its scholarship program partners.

The award of scholarships to promising students and recent graduates pursuing careers in equine medicine is one important way in which The Foundation helps improve the welfare of horses. The scholarships help offset the cost of a veterinary education so that recipients are able to focus on patient care when entering practice.

Scholarships totaling $366,000 were presented prior to the Frank J. Milne State-of-the-Art Lecture on Dec. 9. Below are the check presentation photos with recipients’ names and schools listed underneath. The Foundation for the Horse sincerely thanks its scholarship program partners for their ongoing investment in horse health and the future of the profession.

Three students shared in Coyote Rock Ranch Scholarship Five students received $5,000 Merck Animal Health proceeds of $300,000. From left: Zoë Williams, Michigan Scholarships. From left: Dr. Craig Barnett of Merck Animal State University; Brooke Woodruff of Coyote Rock Ranch; Health; Haydan Vosburgh, Kansas State University; Megan Natalie Andrews, North Carolina State University; The Sitzman, Oklahoma State University; Corey Payne, Texas Foundation for the Horse Chair Dr. Rick Mitchell; and A&M University; Kimberly Hildreth, University of Gabriel Gonzalez, North Carolina State University. Andrews Pennsylvania; Kalie Beckers, Louisiana State University; and received an additional $75,000 award for her designated The Foundation for the Horse Chair Dr. Rick Mitchell. career path of theriogenology.

Five students received $5,000 Oakwood Foundation Four graduates of 2019 received $4,000 Zoetis Scholarships. Scholarships. From left: The Foundation for the Horse From left: Jon Lowe of Zoetis; Dr. Katie Larson, Mississippi Research Subcommittee Chair Dr. Anthony Blikslager; State University; Dr. Rachel Hilliard, Cornell University; Dr. Chelsea Folmar, Texas A&M University; Alexis Daggett, Sheila Spacek, Colorado State University; Dr. Jenny Hamilton University of Wisconsin; Kelsey Palsgaard, University of Virginia-Maryland CVM; and The Foundation for the Horse California, Davis; Lindsay Seewald, Cornell University; The Chair Dr. Rick Mitchell. Foundation for the Horse Chair Dr. Rick Mitchell; and AAEP Past President Dr. Reynolds Cowles, Jr. Not pictured: Ellen Staples, University of Florida. INDUSTRY AAEP News February 2020 XI

US Equestrian implements policy on use of Zimeta™ (dipyrone injection)

US Equestrian on Dec. 31 announced requirements for use of Zimeta™ (dipyrone injection), which was approved in November by the U.S. Food and Drug Administration’s Center for Veterinary Medicine for use in horses for the control of pyrexia (fever).

Dipyrone is a non-steroidal anti-inflammatory drug; however, it is not one of the seven quantitatively restricted NSAIDs currently addressed by USEF Equine Drugs and Medications Rules. As such, it is a prohibited substance, but GR411 (Conditions for Therapeutic Administrations of Prohibited Substances) will apply. Effective immediately, any use of Zimeta will require a properly filed medication report form documenting a 24-hour withdrawal. Additionally, the administration of dipyrone will not constitute the use of a second NSAID, which is prohibited by USEF rules.

For more information, contact the USEF Equine Drugs and Medications Program at (800) 633-2472.

Medication reforms announced for 2-year-old sales

Keeneland, Fasig-Tipton and Ocala Breeders’ Sales No medication may be administered 24 hours prior Company jointly announced significant changes to to a horse’s performance in an under-tack show. The medication policies at the auction companies’ 2-year-old schedule for administration of permitted medication at sales. Effective March 2020: Keeneland’s April Two-Year-Olds in Training Sale will be governed by the rules of racing as set forth by the • No more than one NSAID may be administered. Kentucky Horse Racing Commission. For more, visit Stacking of NSAIDS is prohibited; and https://tinyurl.com/tbred2020. • Bronchodilators (Clenbuterol, Albuterol and all other bronchodilators) are prohibited substances and may not be administered.

AAEP Educational Partner Profile: Kindred Biosciences, Inc.

Kindred Biosciences, Inc., is a publicly traded (NASDAQ: KIN), commercial-stage biopharmaceutical company focused on saving and improving the lives of horses and companion animals. Its mission is to bring to horses and pets the same kinds of safe and effective medicines that human family members enjoy.

In November 2019, KindredBio received FDA approval on its first equine product: Zimeta™ (dipyrone injection). The company also has a deep pipeline of therapeutics in development and is investigating product candidates for the treatment of equine gastric ulcers and equine metabolic syndrome. KindredBio’s drug development program is aimed at fulfilling the immediate need for horse-specific, FDA-approved drugs to treat many common health issues horses—and the veterinarians, owners and trainers who care for them—encounter.

KindredBio is proud to support the veterinary community through its AAEP Educational Partnership, as the Health and Wellness sponsor at the 2019 AAEP Annual Conventions, and through continuing education programs.

For more information about KindredBio, visit kindredbio.com. XII AAEP News February 2020 INDUSTRY

48-hour NSAID administration among model rule amendments adopted by ARCI

At its December meeting in Tucson, Ariz., the Association of Racing Commissioners International (ARCI) approved Palmer 10 amendments to its model rules for racing, including updates to rules pertaining to use of non-steroidal anti- Scott inflammatory drugs (NSAIDs), bisphosphonates and Dr. intra-articular injections.

Board-approved model rule amendments prohibit: • Administration of NSAIDs within 48 hours of a race • Any horse from racing in flat or jump racing within 14 days after receiving an intra-articular injection, and stacking is prohibited • Use of bisphosphonates in any horse younger than four, and the bisphosphonates must be FDA- approved, administered according to instructions and only used to treat navicular disease • Chemical or immunocastration Drug Testing Standards and Practices Committee, which is to present final recommendations in advance of ARCI’s Among other revisions, the board also approved signifi- board meeting in April. cantly increased penalties for “doping” and “equine endangerment” violations pending further work by the Visit arcimodelrules.online for more information.

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Highlights of recent clinically relevant papers

Kissing spines plots supported the agreement between combined and independent testing. The objective of this retrospective study by Amanda Prisk and Combining the TRH stimulation test and the two-step Jose Garcıa-Lopez from Tufts University, USA, was to insulin sensitivity test appears to be a useful diagnostic tool for determine the long-term return to performance of 71 horses equine practitioners in the field, allowing simultaneous testing with impinging and/or overriding dorsal spinous processes of a horse for both PPID and ID. (ORDSP) treated with interspinous ligament desmotomy (ISLD) and to determine the prognostic value of radiographic and scintigraphic grades. Stimulation of subchondral bone cyst healing Following a review of medical records, preoperative In this study, Lance Frazer and colleagues in the United States radiographic and scintigraphic images were evaluated with aimed to predict the bone formation stimulus of a grading scales. Follow-up was obtained with a telephone transcondylar screw across an equine subchondral bone cyst questionnaire. Prognostic value of the number of affected (SBC) in an equine medial femoral condyle (MFC). interspinous spaces, radiographic grading and scintigraphic Finite element modelling (FEM) of an equine MFC with a 2 grading was tested by using a chi-squared test. cm3 SBC under several transcondylar screw conditions was A median follow-up of 35 months was available for 56 performed. horses. Fifty-one of 56 (91.1%) horses returned to some level of The FEM was derived from computed tomography of the performance, with 27/51 (52.9%) horses achieving an right stifle of a yearling Thoroughbred without stifle disease equivalent or higher level of performance. Achieving a lower that had been subjected to euthanasia for reasons unrelated level of performance was attributed to recurrent back pain in to this study. The transcondylar screw was modelled as a 4.5- 9/24 (37.5%) horses and to unassociated lameness in 9/24 mm stainless steel cylinder. The region of interest was the (37.5%) horses. Owners of 44/56 (78.6%) horses were satisfied centrodistal MFC, and bone stimulus was calculated. The with the post-operative performance level, and owners of 46/ stimulus threshold for bone formation (BFT) was >60 MPa, 56 (82.1%) horses would recommend the procedure. None of presented as the percentage of total bone surface area the variables tested influenced the return to performance. (BFA) and frontal plane maps. Principal compressive stress Interspinous ligament desmotomy allowed horses with vectors were also determined. Tested variables were daily ORDSP to return to some level of performance, although cycles, load, and screw compression and position. fewer returned to an equivalent or higher level of At 750 cycles and 900-N load, <3% of the BFA exceeded performance than had previously been reported. the BFT. Increases in BFA > BFT occurred proportionally with load, screw compression and daily cycles (steps). Testing for PPID and insulin dysregulation Compressive stress was oriented vertically on the SBC surface without a screw but aligned with the long axis of well-placed In this study, Remona Horn and Francßois-Rene Bertin from the lag screws. Screw placement through the void also increased University of Queensland, Australia, evaluated combined the number and magnitude of compressive vectors. testing to simultaneously diagnose pituitary pars intermedia This model predicted that a transcondylar lag screw dysfunction (PPID) and insulin dysregulation (ID) in horses. across an MFC SBC increased surface BFA stimulation and The thyrotropin-releasing hormone (TRH) stimulation test reoriented the compression vector. Increasing screw and the two-step insulin sensitivity test are commonly used compression, load and steps per day increased the bone methods to diagnose, respectively, PPID and ID. This study formation stimulus. This study provides evidence that supports investigated the diagnostic value of combining these two the use of a lag screw through an MFC SBC to promote bone tests to diagnose PPID and ID simultaneously. formation. This randomised prospective study included 27 adult horses, 10 control horses without PPID or ID, five horses with PPID only, five horses with ID only and seven horses with PPID Intra-articular injection of antimicrobials and ID. This knowledge summary by Helen Braid from the University of Horses underwent a TRH stimulation test alone, a two-step Liverpool, UK, considers the PICO question ‘In horses, does insulin sensitivity test alone, and combined testing with treatment with intra-articular antimicrobials concurrently with simultaneous TRH and insulin injection in the same syringe. intra-articular corticosteroids reduce the risk of iatrogenic Data were compared by two-way repeated measures synovial sepsis compared to intra-articular corticosteroids analysis of variance and two one-sided tests to demonstrate alone?’ equivalence. Bland–Altman plots were generated to visualise Antimicrobials such as amikacin sulphate or gentamicin agreement between combined and independent testing. are often added to corticosteroid preparations when The effect of combined testing on plasma performing intra-articular injections in horses. This summary adrenocorticotropic hormone, blood glucose concentration or considers whether the risk of iatrogenic synovial sepsis percentage decrease in blood glucose concentration was not increases in the absence of these antimicrobials and whether significantly different from the effect obtained with injecting them concurrently with corticosteroids is necessary. independent testing. One control horse appeared falsely Three studies were examined, two of which directly positive for PPID, two PPID-only horses appeared falsely positive evaluated the prevalence of iatrogenic synovial sepsis for ID and one PPID and ID horse appeared falsely negative for following intra-articular injection. Two of the three studies were ID when tests were performed simultaneously. Bland–Altman

retrospective analyses of medical records, and one was a Medical records from seven referral hospitals in the UK survey-based study with retrospective analyses of medical were reviewed to identify horses that presented with synovial records submitted in response to a questionnaire. contamination/sepsis of the calcaneal bursae between 2002 In this literature, the author found there was no evidence and 2016. A total of 128 horses were included in the study; showing that intra-articular injection of antibiotics in 123 of which recovered from anaesthesia. Following surgery, conjunction with corticosteroids reduces the risk of synovial clinical signs improved in 74.8% (n = 92) of cases. Twenty-one sepsis. However, the intra-articular injection of polysulphated horses underwent a second surgery. Both the calcaneal glycosaminoglycans (PSGAGs) in combination with bursa and the subcutaneous bursa were affected in 42.5% (n corticosteroids was noted as a risk factor for developing = 54) of horses, and in 48.0% (n = 61) only the calcaneal iatrogenic synovial sepsis and therefore concurrent antibiotic bursa was affected. All horses with only the subcutaneous injection when administering PSGAGs may be warranted. The bursa involvement survived. There was bone involvement in reported frequency of infection following intra-articular 23.6% (n = 30) of horses. The most common bacteria isolated injections was very low (0.02–0.08%). An overall prevalence of were Staphylococcus spp., followed by Streptococcus spp. iatrogenic synovial sepsis following all intra-articular injections and Escherichia coli. based on data from all included studies was calculated as Administration of systemic antimicrobials prior to referral 0.02%. However, due to the paucity of literature on the topic, was associated with reduced mortality. Bone fracture/ further studies are required in this field to determine more osteomyelitis, tendon involvement, duration of general accurate clinical recommendations. anaesthesia, post-operative synoviocentesis and post- operative wound dehiscence were associated with increased Atrial fibrillation mortality. At follow-up, 87.1% horses were alive, 7.1% had been euthanised due to the calcaneal injury and 5.7% had This retrospective case series by Ingrid Vernemmen and been euthanised for unrelated reasons. From 57 horses with colleagues in Belgium investigated whether a high atrial athletic performance follow-up, 91.2% returned to the same premature depolarisation (APD) burden after cardioversion is or higher level of exercise. associated with recurrence of atrial fibrillation (AF). Endoscopic treatment of calcaneal bursae Medical records were searched for horses that successfully contamination/sepsis in horses has a good survival rate with underwent transvenous electrical cardioversion (TVEC) for AF the majority of horses returning to their previous athletic use between 2008 and 2017. Horses were included in the study if at the same or higher level. they met the following inclusion criteria: it was their S. WRIGHT first episode of AF, there was no AF recurrence in the first 5 EVE Editorial Office days post-cardioversion, 24-h ECG recording and echocardiographic examination 5 days post-cardioversion were available, no antiarrhythmic treatment was administered during the 24-h ECG recording and follow-up information was available for at least 1 year. An APD was defined as a References premature P-wave, occurring at least 8% earlier, with a normal Braid, H.R. (2019) Does intra-articular injection of antimicrobials or abnormal morphology and, if conducted to the ventricles, alongside corticosteroids or other medications reduce the risk of followed by a QRS-complex with a normal morphology. synovial sepsis? Veterinary Evidence 4,4. Eighty horses met the inclusion criteria, 33% of which (n = Frazer, L.L., Santschi, E.M. and Fischer, K.J. (2019) Stimulation of subchondral bone cyst healing by placement of a transcondylar 26) experienced AF recurrence within 1 year, with a median screw in the equine medial femoral condyle. Vet. Surg. 48, 1194– time to recurrence of 125 days. Horses experiencing 1203. fi recurrence within 1 year showed a signi cantly higher Horn, R. and Bertin, F.R. (2019) Evaluation of combined testing to number (median [range]) of APDs (15 [1–152]) compared simultaneously diagnose pituitary pars intermedia dysfunction and with the non-recurrence group (7 [0–304]). In the multivariable insulin dysregulation in horses. J. Vet. Intern. Med. 33, 2249–2256. model, ≥25 APDs/24 h, mitral regurgitation, left atrial active Isgren, C.M., Salem, S.E., Singer, E.R., Wylie, C.E., Lipreri, G., Graham, fractional area change and lower bodyweight was R.J.T.Y., Bladon, B., Boswell, J.C., Fiske-Jackson, A.R., Mair, T.S. and ı significantly associated with AF recurrence. Rubio-Mart nez, L.M. (2019) A multi-centre cohort study investigating the outcome of synovial contamination or sepsis of These results indicate that the APD burden 5 days post- the calcaneal bursae in horses treated by endoscopic lavage and cardioversion could be a useful predictive value for AF debridement. Equine Vet. J. Epub ahead of print. https://doi.org/ recurrence within 1 year in horses. 10.1111/evj.13180. Prisk, A.J. and Garcıa-Lopez, J.M. (2019) Long-term prognosis for return to athletic function after interspinous ligament desmotomy for Calcaneal bursae sepsis treatment of impinging and overriding dorsal spinous processes in horses: 71 cases (2012–2017). Vet. Surg. 48, 1278–1286. This the UK-based cohort study by Cajsa Isgren and colleagues reports the outcome of horses with Vernemmen, I., De Clercq, D., Decloedt, A., Vera, L., Van Steenkiste, G. and van Loon, G. (2019) Atrial premature depolarisations five contamination/sepsis of the calcaneal bursae following days post electrical cardioversion are related to atrial fibrillation endoscopic treatment in addition to identifying prognostic recurrence risk in horses. Equine Vet. J. Epub ahead of print. factors associated with survival. https://doi.org/10.1111/evj.13186.

Editorial Equine welfare

It is generally accepted that equines benefit from their If we look back to 1992 there were the three fatal association with man. Their use is beneficial. Their abuse is falls during the cross-country phase of Badminton’s, UK, not. Similarly, stress is largely beneficial, but distress is to be 3-day-event, next there was NBC’s one-sided coverage of avoided. The welfare of all animals has become increasingly the 3-day-event at the Olympic Games in Barcelona, which topical. This varies from public rejection of animal appeared to concentrate primarily on horses falling and experimentation, through factory farming, to the stresses which caused subsequent media and public outrage. Then imposed by long journeys, often under deplorable conditions there was the fatal injury sustained by the 8-year-old gelding, with little, if any, food and water which many horses (and Sir Arkay, when he broke a hind leg on the steep, newly built, other animals) must endure on their final travels mostly from bank at Wembley (jumping) Stadium in London, UK and Eastern Europe or from South America, to the slaughter finally Mr Brook’s broken leg in the six-furlong Sprint at the houses in Belgium, France and Italy. There is no problem in Breeders Cup in Hallandale, Florida. This is not to mention the eating horse meat to meet the demand but there can be USA’s Sports Illustrated story of fraud, whereby horses were little justification for not slaughtering them (and other animals) killed to claim the insurance money. None of this did much close to home and so avoid these horrendous journeys. It good for the image of equestrian sport. would be infinitely preferable to have them transported ‘on To add insult to injury, Animals International, the the hook’ rather than ‘on the hoof’. publication for the World Society for the Protection of Let’s look at the developing world. Several UK based Animals, in its winter edition of 1992, grouped together equine charities, in addition to their national work, also photographs and accompanying texts of: operate internationally. World Horse Welfare, for example runs • Clubbing seals to death in Canada; practical training programmes in some of the world’s poorest • Bear-baiting using bull terriers in Pakistan countries, often in conjunction with other welfare charities • A horse falling at the 3-day-event at the Olympic Games and always in partnership with local organisations and in Barcelona, Spain. governments to ensure that their impact is sustainable, to ‘ combat the major causes of equine suffering and thereby The caption referring to the latter read Cross country cruelty ’ helping the owners to help themselves. The aim is to improve at the Olympics and went on to state how horses collapsed the well-being of the equine, be it horse, mule or donkey and from exhaustion and numerous horses were withdrawn or ’ thus improve its working capacity to the benefit of its owner eliminated due to fatigue. In fact, according to the FEI sAnnual and his/her family. World Horse Welfare believes in using Report of 1992, 82 horses representing 18 countries took part in locally available materials to establish sustainable and the 3-day-event at the Barcelona Olympic Games, 72 horses permanent solutions to problems in the world where needed. completed the endurance phase including all 18 teams and 62 In saying this we must always remember the hugely varied horses completed the whole event including 15 teams. Only culture between countries. Religions, history, tribal rivalries, two horses, two too many, presented symptoms of extreme acute poverty and extreme wealth can all influence attitudes exhaustion, both of which fully recovered thanks to excellent ‘ to animals and in some countries the concept of ‘animal veterinary care. The report concluded: This has to be the rights’ or even ‘animal welfare’ may not be recognised. most satisfactory result in the history of the Olympic Games ’ ‘ At home, in the sport horse world, both in racing and in the 3-day-event and had certainly nothing to do with cross ’ competition arena, the specialist equestrian press publishes country cruelty as alleged by Animals International. what they see. It is up to us to ensure that what they see is Resulting from such incidences and to tackle the issues acceptable. A smile and a pat on the neck are infinitely concerned, the FEI, in conjunction with World Horse Welfare reacted by: preferable to a scowl and a jab on the reins from a rider • following an unsuccessful round. In racing, the sight of a horse Forming an Ethics Committee to investigate and monitor welfare issues in equestrian sport, perceived or otherwise; being ridden out to obtain maximum placing, encouraged by • the jockey’s hands and heels and maybe a slap down the Producing a Code of Conduct for the Welfare of the Horse shoulder is surely preferable to the image of raised whips, which was widely circulated in many languages and was flapping reins, with the jockey often seen to be bumping in the updated on a regular basis and published in event schedules and programmes worldwide; saddle. Regardless, this surpasses visions from the past of horses • being truly beaten, particularly in long distance steeplechases Appointing an Honorary Chief Steward, to liaise with and and in heavy ground when they are exhausted and can barely oversee national stewards in its member national federations to standardise the role of stewards in the field; raise a canter. In many countries, but not all, the whips are • now made with softer materials, which cause little or any Introducing Warning Cards (as in football) to be issued fi discomfort. However, this image does not escape the when horses are seen to be abused or when of cials are perception of inflicting pain and injury on the horse, sadly. In either verbally or physically assaulted. the recent past, the winner of the Gold Cup, the major race in What were the main areas of concern in the different the capital city of an African country, could be found 2 years disciplines? later, lame and emaciated, pulling a cart full of rubbish – what • Jumping: primarily wear and tear, locomotion problems, a fall from grace to be discarded by its callous owner, when its pain management, frequency of competition and racing days and fame were over. transport between events;

• Dressage: competition was not considered as stressful as themselves could ever hope to replicate. They considered jumping although the indications were that training this to be the norm and this inevitably resulted in over- methods may well be more so (e.g. Rollkur/Hyperflexion), ridden and exhausted horses or worse, but often without particularly in inexperienced hands; having sufficiently experienced veterinary expertise to • Eventing: tragic deaths of both horses and riders resulted in cope and care for these horses after the ride. It was considerable emphasis being placed on improving safety considered essential to effectively monitor this discipline measures. Most rider deaths had been caused by slow very carefully as it continues to grow, often in countries somersaults and proposals were considered as to how to with little concern for the welfare aspects of their horses; modify various elements of the discipline, hence, Three • Vaulting: although the level of soundness of the horses Day Eventing became Eventing following the removal of concerned often left much to be desired the demands on the steeplechase and the two roads and tracks, prior to the horse were not considered excessive and no specific the 2004 Olympic Games in Athens; welfare problems were known, other than the continuous • Driving: no specific problems; circle on one rein. • Endurance: this was recognised to be a rapidly growing discipline, particularly in the Middle East. The United Arab In conclusion and in the words of World Horse Welfare, Emirates (UAE) led this discipline in the Middle East. It Use but do not abuse our great friend, the horse. promoted and encouraged other countries in the region to organise rides. These countries saw the UAE riders on A. ATOCK good, expensive horses travelling much faster than they Herefordshire, UK

Case Report Theiler’s disease associated with administration of tetanus antitoxin contaminated with nonprimate (equine) hepacivirus and equine parvovirus-hepatitis virus J. J. Kopper†* , H. C. Schott II† , T. J. Divers‡, T. Mullaney§, L. Huang§, E. Noland§ and R. Smedley¶ †Large Animal Clinical Sciences, Michigan State University, East Lansing, Michigan; ‡Clinical Sciences, Cornell University, Ithaca, New York; §Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan; and ¶Veterinary Diagnostic Laboratory, Michigan State University, East Lansing, Michigan, USA *Corresponding author email: [email protected] Dr Kopper's present address: Veterinary Clinical Sciences, Washington State University, Pullman, Washington, USA

Keywords: horse; survival; serum hepatitis; hepatic necrosis; viral hepatitis

Summary were positive for nonprimate (equine) hepacivirus (NPHV) and a novel equine parvovirus-hepatitis virus (EqPV-H). PCR An 11-year-old Trakehner gelding was presented for testing of the lot of tetanus antitoxin administered to the evaluation of lethargy, decreased appetite, mild icterus and gelding 3 months previously also yielded positive results for elevated hepatic enzyme activities. Physical examination, NPHV and EqPV-H. Treatment included supportive care and serum chemistry results and liver biopsy histopathological clinical signs resolved within one week, although hepatic ﬁndings were supportive of Theiler’s disease. Polymerase enzyme activities remained elevated for several months. The chain reaction (PCR) testing results of serum and liver tissue horse successfully returned to work as a hunter/jumper for about one year until it developed a forelimb lameness and progressive atrophy of shoulder musculature (sweeney), prompting a decision for euthanasia 20 months after initial evaluation. Serial PCR testing of serum revealed persistent infection with both NPHV and EqPV-H and necropsy examination revealed chronic active hepatitis, mild liver atrophy and positive PCR results for NPHV and EqPV-H in liver tissue. This case highlights the possible risk of administering potentially contaminated biologics of equine origin and the importance of screening for recently identiﬁed hepatic viruses in donors from which blood products are prepared (Fig 1).

Key points CV Equine parvovirus-hepatitis virus (EqPV-H) is likely the most important cause of Theiler’s disease in horses. Horses can remain persistently infected with EqPV-H after resolution of clinical signs. Donor horses used for production of biological Fig 1: Liver; horse. There is mild centrilobular to scattered products (tetanus and botulinum antitoxins, plasma mononuclear inﬂammation and multifocal hepatocellular necrosis. and stem cells) should be tested annually for EqPV-H. Hepatic cords are disorganised and several hepatocytes are hypereosinophilic, shrunken and have pyknotic nuclei (arrowhead). An individual hypereosinophilic necrotic hepatocyte undergoing karyorrhexis (acidophil body) is indicated by the arrow. CV = central vein. HE 2003.Bar= 150 lm

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Clinical Commentary Theiler’s disease T. J. Divers†* and J. E. Tomlinson‡ †Department of Clinical Sciences, College of Veterinary Medicine, Cornell University; and ‡Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA *Corresponding author email: [email protected]

Keywords: horse; hepatitis; Theiler’s disease; adult horses; viral

Theiler’s disease (equine serum hepatitis) was initially reported of the liver (Robinson et al. 1975). Alzheimer type II cells by Sir Arnold Theiler in 1918 (Theiler 1918). Theiler had characteristic of hyperammonaemia are frequently observed administered pooled equine-origin African horse sickness upon microscopic examination of the brain (Tomlinson et al. (AHS) virus and antiserum to 1148 adult horses as prophylaxis 2018a,b). Horses that survive Theiler’s disease are generally during an AHS outbreak in South Africa. Four to 12 weeks improved within 4–10 days after onset of clinical signs, and following administration of the antiserum, an epidemic of those horses seem to have complete clinical recovery from fulminant hepatic failure occurred in 27 horses that had the disease. To our knowledge, there are no reports of horses received the antiserum. Twenty-two of the 27 horses with that survive Theiler’s disease and then have chronic liver hepatitis died (case fatality rate 81.5%). Theiler also reported failure later in life. In 1995, Guglick reported on two horses that four horses that had not received antiserum but were in with fulminant Theiler’s disease and five horses from the same contact with inoculated horses died with what appeared to farm with subclinical hepatitis, all following administration of be an identical disease, suggesting that a transmissible and TAT 7 and 8 weeks earlier (Guglick et al. 1995). This report contagious pathogen could be responsible for the disease. In indicated that both clinical hepatitis and subclinical hepatitis the United States (US) during the pandemic of western can occur with Theiler’s disease. equine encephalomyelitis (WEE) that began in 1930, findings A summary of these prior publications on Theiler’s disease like those reported by Theiler occurred when pooled equine- suggests the following: (i) clinical hepatitis following origin WEE antiserum was administered to 9193 horses as administration of an equine-origin biologic product is not prophylaxis against WEE infection (Marsh 1937). In that study, common, but when clinical disease occurs, it is often 90 cases of liver failure occurred within 12 weeks following fulminant with onset of clinical signs most commonly administration of the antiserum, with 90% of cases occurring 4–12 weeks following inoculation with an equine-origin blood 40–70 days after the antiserum inoculation. Seventy-nine product; (ii) biologic products produced from pooled serum (87.8%) of the clinically affected horses died within 24 h after might carry a greater risk than single-source blood products; exhibiting neurological signs (hepatic encephalopathy) and (iii) subclinical hepatitis can also occur in horses inoculated jaundice. There are similar outbreaks of hepatic failure with equine-origin biologic products; (iv) horses in contact reported in Europe following administration of equine with inoculated and diseased horses can develop acute antiserum for anthrax or equine influenza (Slagsvold 1938; hepatic disease, suggesting that the disease could be both Thomsett 1971). In the two latter outbreaks, the percentage infectious and contagious. of horses developing liver failure following serum inoculation Since 2012, four new viruses that infect horses (three RNA was low and similar to that previously reported in both the viruses and one DNA virus) have been investigated using South Africa and US outbreaks. The case fatality rates in these unbiased amplification and high-throughput sequencing of four large outbreaks were all very high with deaths most serum and tissue samples from horses. Two of the RNA viruses commonly occurring within 1–5 days following onset of from the pegivirus family, equine pegivirus and Theiler’s clinical signs. disease-associated virus (TDAV), are not thought to be In the last four decades, Theiler’s disease reports have hepatotropic and do not cause liver disease in horses been predominantly limited to scattered case reports within (Chandriani et al. 2013; Kapoor et al. 2013). Theiler’s disease- theUSandtworecentcaseseries(Tomlinsonet al. 2018a,b). associated virus was discovered during the investigation of Reported cases have been most commonly associated with Theiler’s disease outbreak on a single farm, which occurred equine-origin tetanus antitoxin (TAT) administration 40–90 days after administration of equine-origin botulinum antitoxin to earlier, and a small number of cases have occurred following horses. In that study, only RNA viral metagenomics analysis equine plasma administration (Messer and Johnson 1994; was performed (Chandriani et al. 2013). Equine pegivirus is a Aleman et al. 2005). Cases with no history of equine-origin very common infection in horses, while TDAV is uncommon. biologic product administration have also been reported The third recently discovered RNA virus (non-primate (Tomlinson 2018b). All reported cases of Theiler’s disease hepacivirus [NPHV], also called equine hepacivirus or have been in horses that were at least 1 year of age. hepacivirus A) is hepatotropic and causes mild, transient Consistent post-mortem finding among horses that have died biochemically detected hepatic disease in horses (Burbelo of Theiler’s disease includes a collapsed, smaller-than-normal et al. 2012; Pfaender et al. 2015; Scheel et al. 2015). An liver, with microscopic findings of acute centrilobular to mid- immune response to the virus is thought to be associated with zonal massive hepatic necrosis. Periportal hepatocytes are the hepatitis (Pfaender et al. 2015; Ramsay et al. 2015; Scheel less severely affected but sometimes necrotic and often et al. 2015). Although some horses remain persistently contain cytoplasmic vacuoles. A mild-to-moderate lymphocytic/ infected with this virus, there is no substantial evidence of plasmatic infiltrate is frequently seen in the periportal region chronic liver disease in association with a chronic infection.

The latest virus of the recently investigated group of after EqPV-H infection followed by resolution of the hepatitis viruses is a DNA virus named equine parvovirus-hepatitis and persistence of infection. High viral prevalence in some (EqPV-H) (Divers et al. 2018). This virus is hepatotropic and populations of horses might create a diagnostic dilemma produces both clinical and subclinical disease in horses. In a when a horse is found to have both liver disease and is PCR- recent prospective study on Theiler’s disease, EqPV-H was positive for EqPV-H in serum or liver. In those cases, serial found in the serum and/or liver of 18 consecutive cases of serum qPCR testing for EqPV-H could be helpful in Theiler’s disease that occurred 4–12 weeks following determining the significance of the viral infection as a administration of an equine-origin biologic product, with substantial decline in viraemia might be expected during the tetanus antitoxin being the most common product administered time of active hepatitis caused by EqPV-H. (Tomlinson et al. 2018a). In 11 of those cases, either the exact Currently, the USDA is working to eliminate both EqPV-H biologic product administered to the Theiler’s disease case or and NPHV from commercial blood products. In the the same lot number of the product was available for testing, meantime, we would agree with Kopper et al. (2020) that if and all were PCR-positive for EqPV-H. An experimental there is an urgent need for administration of an untested infection study had previously confirmed transmission of EqPV- equine-origin blood product to an adult horse, it should be H from a PCR-positive biologic product to seronegative administered since the incidence of Theiler’s disease is low. horses, proving that transmissible virus (EqPV-H) is present in On the other hand, routine administration of untested equine- some biologic products in the US (Divers et al. 2018). That origin blood products, especially those that are sourced from study also retrospectively tested stored frozen samples (case multiple animals and then pooled, should be administered serum and antitoxin product) from the farm outbreak where only after careful consideration. TDAV had been discovered and found that all samples were also positive for EqPV-H. Author’s declaration of interests Although recent publications demonstrate a strong association of EqPV-H with Theiler’s disease, additional studies No conflicts of interest have been declared. are needed to further confirm EqPV-H as the cause of Theiler’s disease. Because the horse in the case report by Kopper et al. Ethical animal research (2020) was infected with both EqPV-H and NPHV, one might wonder if a co-infection was responsible for the disease in that Not applicable. horse. While co-infection could play a role in the disease, a recently published article found that NPHV infection is rare in Source of funding field cases of Theiler’s disease (Tomlinson et al. 2018a). Additional EqPV-H studies are required to understand the Boehringer Ingelheim Advancement in Equine Research mechanism of the hepatic disease. In experimental EqPV-H Award, Harry M. Zweig Memorial Fund for Equine Research, infections, the onset of biochemical disease correlated with National Institute of Food and Agriculture Grant 2016 and both the time of peak viraemia and initial antibody detection Niarchos Family Equine Research Award. (Divers et al. 2018). It would appear that 4–12 weeks are necessary for these events to occur. The lymphocytic infiltrate often found histologically in the periportal areas may suggest Authorship an acute immune reaction, possibly involving cytotoxic Both authors contributed to the preparation and approval of lymphocytes, towards the virus-laden hepatocytes (Divers this manuscript. et al. 2018). Cases of Theiler’s disease not associated with the recent administration of an equine-origin biologic product are reported, and these cases also have a strong association with References EqPV-H infection (Tomlinson et al. 2018b). Although the Aleman, M., Nieto, J.E., Carr, E.A. and Carlson, G.P. (2005) Serum method of EqPV-H transmission in the ‘non-biologic’ cases is hepatitis associated with commercial plasma transfusion in horses. unknown, it was recently reported that non-biologic cases J. Vet. Intern. Med. 19, 120-122. occur between late spring and late fall, suggesting the Burbelo, P.D., Dubovi, E.J., Simmonds, P., Medina, J.L., Henriquez, J.A., possibility of insect spread of the virus, but this is currently Mishra, N., Wagner, J., Tokarz, R., Cullen, J.M., Iadarola, M.J., Rice, unproven (Tomlinson et al. 2018b). C.M., Lipkin, W.I. and Kapoor, A. (2012) Serology-enabled discovery of genetically diverse hepaciviruses in a new host. J. Infection (EqPV-H) prevalence studies are scarce, but PCR Virol. 86, 6171-6178. testing has detected viral nucleic acid in horses residing in Chandriani, S., Skewes-Cox, P., Zhong, W., Ganem, D.E., Divers, T.J., many US states and in China, and prevalence can be highly Van Blaricum, A.J., Tennant, B.C. and Kistler, A.L. (2013) variable between farms in the same geographic region (Lu Identification of a previously undescribed divergent virus from the et al. 2018). Chronic EqPV-H infections appear to be Flaviviridae family in an outbreak of equine serum hepatitis. Proc. common, and therefore, one might expect EqPV-H infection Natl Acad. Sci. USA 110, E1407-E1415. prevalence to increase with age. One study testing a Divers, T.J., Tennant, B.C., Kumar, A., McDonough, S., Cullen, J.M., random set of serum samples from presumably healthy horses Bhuva, N., Jain, K., Chauhan, L.S., Scheel, T.K.H., Lipkin, W.I., Laverack, M., Trivedi, S., Srinivasa, S., Beard, L., Rice, C.M., Burbelo, reported an EqPV-H nucleic acid prevalence of 13%, and all P.D., Renshaw, R.W., Dubovi, E. and Kapoor, A. (2018) A new virus-positive samples also contained antibody against EqPV- parvovirus associated with serum hepatitis in horses following H, suggesting that none were acute infections (Divers et al. inoculation of a common equine biological. Emerg. Infect. Dis. 24, 2018). All of the EqPV-H-positive horse serum samples in that 303-310. study had normal gamma-glutamyltransferase (GGT) values, Guglick, M.A., MacAllister, C.G., Ely, R.W. and Edwards, W.C. (1995) but it is plausible that these horses might have previously Hepatic disease associated with administration of tetanus antitoxin in eight horses. J. Am. Vet. Med. Assoc. 206, 1737-1740. experienced a subclinical, transient liver disease 4–12 weeks

Kapoor, A., Simmonds, P., Cullen, J.M., Scheel, T.K.H., Medina, J.L., Scheel, T.K.H., Kapoor, A., Nishiuchi, E., Brock, K.V., Yu, Y., Andrus, L., Giannitti, F., Nishiuchi, E., Brock, K.V., Burbelo, P.D., Rice, C.M. and Gu, M., Renshaw, R.W., Dubovi, E.J., McDonough, S.P., Van de Lipkin, W.I. (2013) Identiﬁcation of pegivirus (GB virus-like virus) that Walle, G.R., Lipkin, W.I., Divers, T.J., Tennant, B.C. and Rice, C.M. infects horses. J. Virol. 87, 7185-7190. (2015) Characterization of nonprimate hepacivirus and Kopper, J.J., Schott II, H.C., Divers, T.J., Mullaney, T., Huang, L., construction of a functional molecular clone. Proc. Natl Acad. Sci. Noland, E. and Smedley, R. (2020) Theiler’s disease associated USA 112, 2192-2197. with administration of tetanus antitoxin contaminated with non- Slagsvold, L. (1938) Occurrences of icterus in horses after inoculation primate (equine) hepacivirus and equine parvovirus-hepatitis of anthrax antiserum. Norsk. Vet. 50, 69. virus. Equine Vet. Educ. 32, 62. https://doi.org/10.1111/eve.12999. Theiler, A. (1918) Acute liver-atrophy and parenchymatous hepatitis in Lu, G., Sun, L., Ou, J., Xu, H., Wu, L. and Li, S. (2018) Identiﬁcation and horses. In: Union of South Africa. Department of Agriculture, 5th genetic characterization of a novel parvovirus associated with and 6th Reports of the Director of Veterinary Research, pp 7-164. serum hepatitis in horses in China. Emerg. Microbes Infect. 7, 170. Thomsett, L.R. (1971) Acute hepatic failure in the horse. Equine Vet. J. Marsh, H. (1937) Losses of undetermined cause following an outbreak 3, 15-19. of equine encephalomyelitis. J. Am. Vet. Med. Assoc. 91, 88-93. Tomlinson, J.E., Kapoor, A., Kumar, A., Tennant, B.C., Laverack, M.A., Messer, N.T. and Johnson, P.J. (1994) Idiopathic acute hepatic disease in Beard, L., Delph, K., Davis, E., Schott li, H., Lascola, K., Holbrook, horses: 12 cases (1982-1992). J. Am. Vet. Med. Assoc. 204, 1934-1937. T.C., Johnson, P., Taylor, S.D., McKenzie, E., Carter-Arnold, J., Setlakwe, E., Fultz, L., Brakenhoff, J., Ruby, R., Trivedi, S., Van de Pfaender, S., Cavalleri, J.M., Walter, S., Doerrbecker, J., Campana, B., Walle, G.R., Renshaw, R.W., Dubovi, E.J. and Divers, T.J. (2018a) Brown, R.J., Burbelo, P.D., Postel, A., Hahn, K., Anggakusuma, Riebesehl, N., Baumgartner,€ W., Becher, P., Heim, M.H., Viral testing of 18 consecutive cases of equine serum hepatitis: a prospective study (2014-2018). J. Vet. Intern. Med. (Dec. 5) (Epub Pietschmann, T., Feige, K. and Steinmann, E. (2015) Clinical course of infection and viral tissue tropism of hepatitis C virus-like ahead of print). nonprimate hepaciviruses in horses. Hepatology 61, 447-459. Tomlinson, J.E., Tennant, B.C., Struzyna, A., Mrad, D., Browne, N., € Ramsay, J.D., Evanoff, R., Wilkinson, T.E., Divers, T.J., Knowles, D.P. and Whelchel, D., Johnson, P.J., Jamieson, C., Lohr, C.V., Bildfell, R., McKenzie, E.C., Laverack, M., Renshaw, R.W., Dubovi, E., Kapoor, Mealey, R.H. (2015) Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus. Hepatology A., Meirs, R.S., Belgrave, R., Engiles, J., Van de Walle, G.R. and ’ 61, 1533-1546. Divers, T.J. (2018b) Viral testing of 10 cases of Theiler s disease and 37 in-contact horses in the absence of equine biologic product Robinson, M., Gopinath, C. and Hughes, D.L. (1975) Histopathology of administration: a prospective study (2014-2018). J. Vet. Intern. Med. acute hepatitis in the horse. J. Comp. Pathol. 85, 111-118. (Dec 5) (Epub ahead of print).

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Case Report Locally invasive melanoma in the internal laminae of the hoof of a bay mule G. Kay†* , P. McNeil‡, D. Knottenbelt§ and N. Tligui¶ †American Fondouk, Fez, Morocco; ‡Department of Veterinary Pathology, University of Glasgow; §School of Veterinary Medicine, Weipers Centre Equine Hospital, University of Glasgow, Glasgow, UK; and ¶Department of Veterinary Pathology, Institut Agricole et Veterinaire Hassan11, Rabat, Morocco *Corresponding author email: [email protected]

Keywords: horse; melanoma; mule; hoof; internal lamina; bay

Summary An 8-year-old bay mule was presented with moderate right forelimb lameness and dark discolouration of the white line. Histopathology of the dark material indicated a melanoma. Extensive resection and debridement were not curative and the mule was subjected to euthanasia 70 days after presentation.

Introduction Melanoma is a common potentially infiltrative neoplasm of equids that is most prevalent in grey individuals. Controversy exists surrounding the nature and aetiology of melanoma in equids and whether they should be classified as benign or malignant (Floyd 2003). It is, however, widely accepted that the condition affects defined predilection sites including the perineum and perianal regions, the base and undersurface of the tail, the lips and the eyelids. Internal structures that are most often involved include the perirectal and parotid Fig 1: Obvious discolouration of white line at presentation. salivary gland regions. Melanoma affecting the foot is extremely rare; only three other cases in horses are recorded in the literature (Kunze et al. 1986; Honnas et al. 1990; Floyd 2003). None of the previous reports describe laminal melanoma, and there are no reports of melanoma affecting the laminae of a horse’s foot or the foot of a mule.

Case history A bay 8-year-old mule was presented with a history of insidious onset moderate right forelimb lameness that had been present for 1 month. The mule was bright and alert, in good body condition, and all other clinical parameters were unremarkable. Lameness examination revealed a 4/5 lameness (AAEP lameness score) of the right forelimb. Examination of the right forelimb limb showed a variable dark discolouration of the white line around the entire circumference of the sole (Fig 1). No draining tracts were present and hoof conformation showed the slightly boxy upright conformation that is common in mules. No difference in hoof conformation was noted between the two forelimbs. Application of hoof testers elicited a mild response without deformation of the solar surface. No digital pulses were palpable. An abaxial sesamoid nerve block was performed using 1.5 mL lidocaine (Lurocaine) and the lameness was abolished. Nothing remarkable was detected on standard Fig 2: Lateromedial radiograph of the right front hoof following radiographic projections (Fig 2). initial debridement and foot preparation.

Examination of the solar surface of the white line with a hoof knife showed the discoloured areas to have an abnormal moist and soft consistency in comparison to the normal parts of the white line. The normal morphology of the internal lamellae was obliterated, and there was no integrity or strength in the solar aspect of the white line.

Clinical course Extensive debridement of the discoloured areas was carried out under sedation and bilateral abaxial sesamoid regional nerve block. A hoof knife and a curette were used to remove as much of the discoloured tissue as possible using a solar surface approach (Fig 3). A tourniquet was used to limit haemorrhage. The discoloured tissue was all black, soft consistency, well deﬁned and restricted to the internal lamellae and did not appear to affect the external lamellar portion of the white line, giving a somewhat ‘zebra striped’ Fig 4: Heart bar shoe in place after initial debridement. appearance to the white line which can be seen in Figure 1. An impression smear of this tissue showed multiple black melanocytes suggestive of a diagnosis of melanoma. A single temporary protective dose of 1500 IU tetanus antitoxin was administered since the animal had no previous vaccination history. The hoof was bandaged after debridement, and thereafter, the defect was cleaned daily with a 0.9% saline solution and re-dressed. Ten days after initial debridement, the defects in the white line appeared to be granulating well with no evidence of discolouration. A heart bar shoe was placed to stabilise the foot (Fig 4) and a hoof boot placed over this to keep the area clean. No change was noted in the degree of lameness. Three weeks after initial debridement, the solar defects were ﬁlling in well with healthy looking, keratinising granulation tissue. However, some exudate appeared to be present deep to the granulating wound, and on further exploration, it was clear that further radical debridement and hoof wall resection were necessary (Fig 5). After performing Fig 5: Hoof after resection of part of the lateral hoof wall showing abnormal tissue deep to the healthy looking granulation tissue.

an abaxial sesamoid nerve block and applying a tourniquet, the hoof wall was resected around the toe and lateral aspect of the hoof up to 1 cm from the coronary band. The abnormal black tissue deep to the hoof wall was further debrided and submitted for histopathology. It was, however, not possible to reach the full extent of the abnormal tissue without removing the entire hoof wall up to the coronary band. Discoloured tissue was still evident on the lateral, medial and dorsal aspects of the sole in all the areas that had been previously debrided. The option of using intralesional cisplatin was discussed at this time to try to improve the prognosis for this animal. The authors, however, decided that intralesional treatment with cisplatin within the hoof capsule was likely to result in severe inﬂammation and further compromise to the integrity of the lamellae. The defect was bandaged and regional perfusion with 1 g of ceftiofur (Ceftiﬂex1) was performed at 48 h intervals for 6 days to prevent secondary infection of the wound. The heart bar shoe was replaced to try to provide additional support to the remaining hoof wall and to redistribute the load to the Fig 3: Solar surface of hoof after initial debridement. palmar aspect of the sole. The lateral hoof wall defect made

nailing difﬁcult; however, by using a combination of a single a) nail in the heel region on the lateral side plus two nails on the medial toe region, the farrier was able to secure the shoe. Despite this attempt at stabilisation and treatment with phenylbutazone (2.2 mg/kg bwt q. 8 h) (Eq Zona2), the mule remained severely lame. Four weeks after wall resection and 8 weeks after presentation, the mule continued to show no improvement in comfort level. Discussions with the owner were initiated to introduce the prospect of euthanasia on humane grounds. In Morocco, the economic value of a good mule to its owner cannot be overestimated. Mules are used to plough ﬁelds, harvest and thresh corn and to transport goods to market. The loss of a mule can represent an economic catastrophe, and therefore, euthanasia is an option that is rarely accepted readily. Several weeks of negotiation were required before the owner could accept the poor prognosis at which point the animal was subjected b) to euthanasia.

Post-mortem findings Post-mortem examination revealed extensive tumour infiltration of the internal lamellae and a small area of 20 μm infiltration of the solar margin of the coffin bone which showed areas of black discolouration (Fig 6). No other evidence of discoloured tissue was found in any other organ system despite a post-mortem examination of all internal organs and skin or in any other region of the limb. Three separate samples of the tumour tissue from the internal lamellae were submitted for histopathology. No tissue from any other organ system was submitted. The samples revealed aggregates and sheets of large, heavily pigmented 200 μm cells, irregular trabeculae and anastomosing strands of Fig 7: a) Heavily pigmented cells form aggregates and sheets stratified squamous epithelium that was often heavily between epithelial lamellae and infiltrate adjacent connective keratinised (Fig 7a). Part of the surface of the sample tissue (top left). [Cracks within the section are due to fixation and comprised a thick layer of parakeratotic keratin surmounting processing artefact]. b) A bleached section reveals large a thick epithelium; pigmented cells and/or clumps of polygonal (ballooned) cells filled with fine yellow-brown granules. pigment granules were scattered through all the layers and High power view (inset) Nuclei are mostly narrow deeply staining crescents but occasional larger irregular structures are seen (top were present in subjacent fibrous connective tissue. left) that may or may not represent mitotic figures. [Correction Evaluation of a bleached section revealed ballooned added on 20 Jun 2018, after first online publication: Figure 7 was previously incorrect and has been revised to 7a and 7b in this version. Citations corresponding to the figures have been updated to reflect this change.

melanocytes filled with fine yellow-brown granules (Fig 7b). Occasional ovoid or irregular nuclei were found in these cells, but the majority appeared as narrow, deeply staining crescents. No mitoses were identified. The histological features suggested a diagnosis of benign melanoma (melanocytoma) although histological appearance is not always a reliable indicator of the clinical behaviour of melanomas at any site.

Discussion This is the ﬁrst report of a melanoma in the hoof of a mule and the ﬁrst report of a locally invasive melanocytoma in the hoof of an equid. All three previous reports of melanomas in the feet of horses involved malignant melanoma that Fig 6: Transverse section of the last phalanx at post-mortem presented with draining tracts and/or obvious hoof wall showing black tissue at the apex of the phalange. defects; they were found on necropsy to have metastasised

extensively within the digit and the limb. In contrast, the reported by Valentine 1995 related to 53 horses. In 2009, 20 present case showed a discolouration of the white line but horses with melanomas were reviewed by Schoniger€ and no draining tract. The lesion was localised and Summers (2009). None of these case series included any mules nonencapsulated. No metastases were evident post-mortem or donkeys. Both previous mule reports refer to single cases of and the histopathology reported characteristics typical of a malignant melanoma in grey mules (Diez 1973; Guiffre et al. benign melanocytoma. Current thinking, however, is that all 1995). Melanoma appears to be rarer still in donkeys, although melanomas in horses and probably other equids are a single case of a malignant melanoma in a grey donkey has potentially malignant, although in grey horses, they may be recently been reported (Mosbah and Awadin 2017). A recent quiescent for some considerable time (Moore et al. 2013). survey of neoplasia in 125 donkeys reported only three of 125 In this case, the local infiltration seems to extend into the animals had melanomas (Davis et al. 2016); none of these bone and this can be construed as malignant behaviour. were found in the ‘usual’ equine predilection sites. The Donkey Cutaneous melanocytic tumours have been classified by Sanctuary necropsy study on 1444 aged donkeys reported not Valentine (1995) into four clinical syndromes, some of which a single melanoma (Morrow et al. 2011). Additionally, only may or may not present a spectrum of pathology. These are one donkey with a melanoma has ever been treated at the as follows: Donkey Sanctuary in the last 10 years (A. Theiman, personal 1 Melanocytic naevus or melanocytoma is more often communication, 2017). Davis et al. (2016) noted that whilst found in younger horses which may be grey or non-grey sarcoid and some other skin tumours are relatively common in and which are localised and nonencapsulated. They are donkeys, melanoma is extremely rare and they speculated considered benign and significant features on that melanoma in horses is possibly associated with an histopathology include a low-mitotic index (<1 phpf) with autosomal dominant trait that may have developed as an heavy melanin pigmentation. unintended consequence of human selection for colour. They 2 Discrete dermal melanoma is generally found in older grey suggest further that the rarity of melanoma in donkeys is a horses with single nodules in predilection sites. These may result of the lack of any significant colour selection in this have a high-mitotic rate but are considered benign whilst species. This case is the first report of a benign melanocytoma having the potential for malignancy. Histopathologically, in the hoof of any equid species. they have a variable mitotic index and they may be more It has been suggested that extensive and complete or less pigmented. resection of benign melanocytoma is curative (Knottenbelt 3 Dermal melanomatosis may represent a spectrum of the 2009), but despite repeated attempts at resection in this disorder which starts as a discrete dermal melanoma and case, it was not possible to remove all of the affected tissue. progresses to involve multiple and coalescent nodules. The tumour showed very little localisation and invaded They are associated with the development of visceral extensively between the hoof and the pedal bone. Previous metastasis; but histologically, they are indistinguishable reports of melanoma in the hoof describe treatment with from discrete dermal melanomas. aggressive surgical resection even including amputation of 4 Malignant melanoma generally affects middle aged to the entire distal limb (Floyd 2003) in a failed attempt to older horses of grey or non-grey colour. These tumours salvage a valuable brood mare. The only other report in the have a high-mitotic index and show aggressive literature of treating a hoof melanoma described surgical widespread metastatic behaviour. resection followed by intralesional treatment with cisplatin (Honnas et al. 1990). This approach appeared to slow The case reported here had a bay coat colour, had only a tumour growth for a further 2 years after presentation at single lesion in a nontypical location, and showed no which point the tumour recurred and the horse was detectable evidence of metastatic activity. On subjected to euthanasia. Obviously, the breeding potential histopathology, no mitotic activity was reported and the of our animal was limited by the fact that it was a mule and tissue was heavily pigmented and therefore was considered its only value was as a working animal. Achieving ‘pasture to be well differentiated. All these features are suggestive of soundness’ would not, therefore, have constituted a success. a benign melanocytoma and are consistent with Valentine Under the prevailing circumstances, there are considerable (1995) description of this classification. As previously cultural, religious, economic and social constraints on mentioned, however, post-mortem examination showed euthanasia, so whilst the delay could be seen as undue, some evidence of infiltration of the third phalanx which due care had to be taken in making the final decision would not normally be consistent with a benign tumour. The acceptable. effect of tumour proliferation within the confines of a hoof Chemotherapy with the use of cisplatin is relatively well capsule being constrained by the hoof wall and the supported by evidence in the literature and reduction in underlying bone might cause significant compromise to one tumour size can be expected, particularly where small or both of these structures. It is possible that the invasion of discrete nodules are involved. A study of 13 horses with the pedal bone by the tumour represented pressure necrosis dermal melanotic tumours treated intralesionally with cisplatin of the bone and tumour invasion at the same time. This might in a sesame oil-based slow-release carrier reported a success have been at least a part of the pain associated with the rate of 81% (Theon et al. 2007). This approach was not tumour. This would be an unusual event for a benign tumour considered to be a viable option in this case because (i) the of this type, but the location of the tumour and its extent material could not be effectively distributed and (ii) any suggests that it can arise and again such a circumstance has resulting inflammation within the hoof capsule would have not to our knowledge been reported. further compromised the already affected laminae. In This case was unusual not only in the location of the addition, cisplatin is a highly toxic compound with significant tumour and the coat colour but also the species. Only two carcinogenic potential to humans; a risk benefit analysis, reports of melanomas in mules exist in the literature. The study therefore, precluded the use of cisplatin in this case.

Authors’ declaration of interests Diez, E.A. (1973) A case of malignant melanoma in mule. Anales de la Facultad de Veterinaria de Leon. http://aims.fao.org/serials/c_9ab No conflicts of interest have been declared. 7e4c Floyd, A.E. (2003) Malignant melanoma in the foot of a bay horse. Equine Vet. Educ. 15, 295-297. Ethical animal research Guiffre, G., Muscarella, A., Muscara, M., Todaro, P. and Tuccari, G. No research undertaken. (1995) Malignant hypomelanotic melanoma of mules. A histologic, immunohistochemical and ultrastructural study of a case with unknown primary site and widespread metastases. Pathologica 87, Source of funding 100-104. Honnas, C.M., Liskey, C.C., Meagher, D.M., Brown, D. and Luck, E.E. None. (1990) Malignant melanoma in the foot of a horse. J. Am. Vet. Med. Assoc. 197, 756-758. Acknowledgements Knottenbelt, D. (2009) Neoplastic conditions. In: Pascoe’s Principles and Practice of Equine Dermatology. Eds: D.C. Knottenbelt and The authors are grateful to Dr Pauline McNeil and Prof J.W. McGarry. Saunders Elsevier, Philadelphia. p 421. Noursaid Tligui for the histopathological assessments and the Kunze, D.J., Monticello, T.M., Jakob, T.P. and Crane, S. (1986) associated photomicrographs. Malignant melanoma of the coronary band in a horse. J. Am. Vet. Med. Assoc. 188, 297-298. Moore, J.S., Shaw, C., Shaw, E., Buechner-Maxwell, V., Scarratt, W.K., Authorship Crisman, M., Furr, M. and Robertson, J. (2013) Melanoma in horses – current perspectives. Equine Vet. Educ. 25, 144-151. This is a case report. D. Knottenbelt, G. Kay, P. McNeil and N. Morrow, L.D., Smith, K.C., Piercy, R.J., du Toit, N., Burden, F.A., Olmos, Tligui all contributed to preparation and approval of the G., Gregory, N.G. and Verheyen, K.L.P. (2011) Retrospective manuscript. analysis of post-mortem findings in 1,444, aged donkeys. J. Comp. Pathol. 144(2–3), 146-156. Mosbah, E. and Awadin, W. (2017) Diagnosis and management of a Manufacturers' addresses malignant dermal melanoma in a donkey (Equus asinus). J. Vet. 1Med Pharmex Inc, Pomona, California, USA. Med. Surg. 1, 1. https://doi.org/10.4172/2574-2868. 2 Lab Calier, 48 Lotissement Azzahra, Oulja, Morocco. Schoniger,€ S. and Summers, B. (2009) Equine skin tumours in 20 horses resembling three variants of human melanocytic naevi. Vet. Dermatol. 20, 165-173. References Theon, A.P., Magdesian, K.G. and Snyder, J.R. (2007) Long term outcome associated with intratumoural chemotherapy with Davis, C.R., Valentine, B.A., Gordon, E., McDonough, S.P., Schaffer, cisplatin for cutaneous tumours in equidae: 573 cases (1995–2004). P.A., Allen, A.L. and Pesavento, P. (2016) Neoplasia in 125 donkeys J. Am. Vet. Med. Assoc. 230, 1506-1513. (Equus asinus): literature review and a survey of five veterinary schools in the United States and Canada. J. Vet. Diagn. Invest. 28, Valentine, B.A. (1995) Equine melanocytic tumours: a retrospective 662-670. study of 53 horses (1988 to 1991). J. Vet. Int. Med. 9, 291-297.

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Case Report Treatment of haemoperitoneum secondary to ruptured granulosa cell tumours in two mares F. C. F. Worsman†1* , S. Z. Barakzai‡2 , M. P. de Bont†3 , S. Turner‡ and L. M. Rubio-Martınez† †Philip Leverhulme Equine Hospital, Institute of Veterinary Science, University of Liverpool, Leahurst, UK; and ‡Chine House Veterinary Hospital, Sileby, Leicestershire, UK *Corresponding author email: [email protected] Present addresses: 1Dick Vet. Equine Hospital, R(D)SVS, Roslin, Midlothian, UK; 2Equine Surgical Referrals, Cheam, Surrey, UK; 3Dierenkliniek De Bosdreef, Moerbeke-Waas, Belgium

Keywords: horse; granulosa cell tumour; ovariectomy; haemoperitoneum; autologous blood transfusion; abdominocentesis

Summary two mares presenting with severe HP secondary to GCTs This report describes two cases of successful surgical which were successfully treated by ovariectomy. management of granulosa cell tumours (GCT) in mares presenting with haemoperitoneum (HP). Controlled Case 1 details abdominal drainage was initially attempted in Case 1 but was not successful. A ventral midline exploratory laparotomy A 19-year-old (272 kg) Pony mare used for driving was allowed removal of a haemorrhaging 13 kg GCT. The mare referred with a history of abdominal distention, inappetance, made a full recovery and returned to normal work as a dullness and lethargy of 1 month duration. The mare was driving pony 11 months post-operatively. In Case 2 controlled reported to have recently displayed abnormal oestrus cycle abdominal drainage was followed by standing left flank behaviour. laparoscopic visualisation of the bleeding ovary and transection of the ovarian pedicle by electrocautery. The GCT Clinical investigations was then removed via a ventral midline incision due to its On presentation at the hospital the mare was quiet, with large size. Haemoperitoneum can be associated with GCTs marked abdominal distention. She was tachycardic and in some cases is severe enough to prompt emergency (96 beats/min) and had weak peripheral pulses. The rectal treatment. Stabilisation of the patient and removal of the temperature was lower than normal (36.2°C). Mucous haemorrhaging GCT can lead to a successful outcome. membranes were very pale with a capillary refill time of 3 s. The mare’s peripheral blood packed cell volume was 11%, the red blood cell count was 2.05 9 1012 cells/L (reference range 6.8–12.9 9 1012 cells/L), total protein was 50 g/L and > Introduction lactate was 9 mmol/L. Percutaneous abdominal ultrasonography with a GETM1 Haemoperitoneum (HP) is uncommon in horses (Dechant 4C-RS probe (2.0–5.0 Hz 60 mm radius convex probe) et al. 2006) and its investigation can be difficult owing to the revealed large areas of mixed echogenicity with a free array of causes and the nature of their manifestation swirling appearance (Fig 1) consistent with HP (Harper et al. (Dechant et al. 2006; Conwell et al. 2010). Causes of HP in 2010). A large mass of mixed echogenicity (Fig 2) which had horses include splenic, hepatic or uterine rupture, broad a multilocular ‘honeycomb’ appearance typical of a GCT ligament haemorrhage, ovarian haematomas, abdominal (White and Allen 1985) was identified. The mass was neoplasia (primary or metastatic), rupture of mesenteric approximately 20 9 30 cm in size and was visible from both arteries secondary to strongyle migration, as well as vascular the left and right paralumbar fossae. Abdominocentesis leakage associated with the renal artery, ileocaecal artery yielded haemorrhagic fluid with a packed cell volume of and aorta (Dechant et al. 2006; Conwell et al. 2010). 40%, a total protein of 56 g/L and total white blood cell Ovarian tumours account for 2.5% of all neoplasms in the count 12 9 109 cells/L which confirmed the presence of a HP. horse (Sundberg et al. 1977) and granulosa cell tumour (GCT) Per rectum examination was attempted but the mare is the most common neoplasm affecting the equine ovary resented the procedure. Following administration of hyoscine (McCue et al. 2006). The most common clinical signs butylbromide (0.3 mg/kg bwt i.v., Buscopan2) to facilitate associated with ovarian GCTs are changes in behaviour, per rectum examination, the mare collapsed and thus per specifically developing aggressive, stallion-like or rectum examination was not completed. nymphomania behaviour, infertility, anoestrous or colic Emergency i.v. fluid resuscitation was initiated. The pony (Crabtree 2011). It is uncommon that mares with GCTs received a 10 L bolus of isotonic crystalloid solution present with secondary HP, and only seven cases have been (Aquapharm 11, solution for infusion, compound sodium previously reported in the literature (Green et al. 1988; lactate i.v. infusion BP3) and an allogenic blood transfusion of Gatewood et al. 1990; Alexander et al. 2004; Dechant et al. 8 L of fresh whole blood (not cross matched) from two 2006; Harper et al. 2010) with only five of those surviving. This hospital donor mares. The mare’s condition stabilised and she case report describes the clinical signs and management of was standing again by approximately 10 min following fluid

© 2018 The Authors Equine Veterinary Education published by John Wiley& Sons Ltd on behalf of British Equine Veterinary Association This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made. 72 EQUINE VETERINARY EDUCATION / AE / FEBRUARY 2020

administered flunixin meglumine (1.1 mg/kg bwt i.v. b.i.d., Meflosyl4) and ceftiofur (2.2 mg/kg bwt i.m. b.i.d., Excenel4) LEFT FLANK pre-operatively. A cephalosporin was chosen over the standard hospital protocol of a penicillin and aminoglycoside to provide a broad spectrum of cover and minimise any nephrotoxic effects due to the hypovolaemic state of the pony. Following induction of general anaesthesia, the mare was positioned in dorsal recumbency for exploratory laparotomy and the ventral abdomen was aseptically prepared for surgery. A 25 cm ventral midline laparotomy incision was made from the umbilicus cranially. Following digital penetration of the peritoneum a substantial volume of sanguinous fluid (approximately 9 L) containing blood clots was spontaneously released. Suction was instigated to aid visualisation within the abdominal cavity. A large, 40 9 30 9 25 cm mass with a firm, multi-nodular texture was palpable, occupying much of the caudal abdomen. Partial Fig 1: Transcutaneous abdominal ultrasound image from Case 1. exteriorisation of the mass revealed the most likely source of Large areas of mixed echogenicity with a free swirling fluid profuse haemorrhage to be a capsular tear. The mass was appearance, consistent with haemoperitoneum. noted to be associated with the right ovary. The right ovarian pedicle was ligated with a combination of transfixing, circumferential ligatures (4M braided-lactomer [Polysorb]5) RIGHT and electrocautery applied with a LigasureTM Vessel Sealing System (Ligasure Atlas5). The 13 kg mass was removed (Fig 3). The abdominal cavity was lavaged with 15 L of sterile polyionic fluid, and as much fluid as possible was then removed with suction post-lavage. No other abnormal findings were found intra-abdominally and the laparotomy incision was closed with a routine three-layer closure. Intraoperatively the mare received a 6 L fresh whole blood transfusion (un-matched) from two hospital donor mares, 1 L bolus of colloids (Geloplasma6), isotonic crystalloid solution (4 mL/kg/h Aquapharm 113) and 1 L bolus of 7.2% sodium chloride hypertonic saline (Vetivex 207). The mare recovered uneventfully from general anaesthesia and isotonic crystalloid i.v. fluid therapy (4 mL/kg/h Aquapharm 113) was continued for 24 h post-operatively. Histopathological examination of the tissue sections from the mass confirmed the diagnosis of a macrofollicular granulosa cell tumour. Fig 2: Case 1: Transcutaneous abdominal ultrasound image via the right paralumbar fossa. A large ovarian mass of mixed echogenicity with hypoechoic cystic structures (black arrows) Post-operative progress adjacent to homogeneous soft tissue stroma consistent with a Post-operative analgesia was provided by administration of multilocular ‘honeycomb’ appearance typical of a GCT. Red flunixin meglumine (0.5 mg/kg bwt i.v. b.i.d., Meflosyl4) for arrows delineate the border of the mass. 5 days. Post-operative antimicrobial therapy consisted of ceftiofur (2.2 mg/kg bwt i.m. b.i.d., Excenel4) for 5 days. and blood administration. Controlled blood collection from The mare showed steady improvement and was the abdominal cavity was attempted following discharged 13 days following surgery. At discharge the abdominocentesis with a metal 14 gauge 5 cm teat packed cell volume was 17%, total protein was 65 g/L and cannula; however, the procedure was aborted due to poor systemic lactate was 0.8 mmol/L. A follow-up phone call with rate of fluid collection and repeated occlusion of the the owner 11 months post-operatively revealed the mare to cannula. The mare’s parameters improved over an hour be doing well and back in work as a driving pony. following blood transfusion and fluid resuscitation; HR 68 beats/min, PCV 16% and pale pink mucous membranes. Case 2 details Owing to the appearance of an abnormal ovary and history of abnormal behaviour, GCT or ovarian haematoma A 10-year-old Thoroughbred mare was presented for were suspected. Although other sources of HP could not be investigation of increasing abdominal distention which had ruled out, a working diagnosis of severe HP possibly been noted over the previous 4 weeks. The mare was associated with a suspected GCT was made. reported to be more aggressive than usual but the owners were unsure of any changes to her oestrus cycle. Clinical Treatment examination revealed a normal heart rate, rectal Owing to the fatal risk of ongoing haemorrhage, emergency temperature and respiratory rate but poor body condition exploratory laparotomy was performed. The mare was with an enlarged abdomen.

Gen MASS

OVARY

Fig 4: Case 2: Ultrasound image obtained per rectum of the ovarian mass with hypoechoic cystic structures and more homogeneous soft tissue stroma within it. Red arrows delineate the dorsal border of the mass, the excessive volume of hypoechoic peritoneal ﬂuid (blue arrow) is also visible. SI = small intestinal loop.

Fig 3: Case 1: following mass removal, an incision was made into the GCT (white arrow) revealing haemorrhage within the mass associated with the capsule rupture. rate appeared to decrease to a slow drip; ultrasound examination was not repeated. After a 24-h period of starvation, the mare underwent Investigations laparoscopic surgery under standing sedation with romifidine Examination per rectum revealed a large (circa 40 cm (0.08 mg/kg bwt i.v., Sedivet2), plus butorphanol (0.01 mg/kg diameter), smooth, hard mass in the right ventral abdomen, bwt i.v., Torbugesic4). Perioperative antibiotics and NSAIDs thought to be the right ovary. All aspects of the mass could were administered 2 h before surgery (gentamicin 6.6 mg/kg not be palpated per rectum due to its size. Ultrasound bwt i.v. s.i.d., Genta Equine7, procaine penicillin 15 mg/kg examination findings per rectum revealed the mass to be bwt i.m. b.i.d., Depocillin8, flunixin meglumine 1.1 mg/kg bwt of heterogeneous echogenicity with multiple areas of i.v., Flunixin9). Portals were made in a triangular formation. The hypoechoic fluid contained within honeycomb-like soft tissue laparoscope portal was made at the level of the ventral stroma and additional areas of homogeneous soft tissue aspect of the right tuber coxa, immediately caudal to the echogenicity (Fig 4) surrounded by an excessive amount of last rib. The second (LigasureTM) portal was made at the same hypoechoic peritoneal fluid. Percutaneous ultrasonographic level dorso-ventrally, but 10 cm caudal to the laparoscope examination over the right flank region and ventral abdomen portal. The third (instrument) portal was made 10 cm confirmed a markedly increased amount of peritoneal fluid vertically below the laparoscope portal. All three portal sites and a similar poly-cystic structure within the mass. were anaesthetised using direct infiltration of 20 mL 2% Routine haematology revealed mild anaemia (RBC lignocaine (Lidocaine injection10) at each site and 12 mm 6.0 9 1012 cells/L, HCT 27.7%, HGB 10.1 g/dL), and mild diameter laparoscope trocars (VersaportTM V² 5mm–12 mm leukopenia (5.3 9 109/L) with lymphopenia (1.22 9 109 cells/L). Long Trocar with Fixation Cannula and Universal Seal5) were Needle centesis of the peritoneal cavity was performed from inserted in a standard fashion (Lloyd et al. 2007). the ventral midline using a 19 gauge 38 mm needle. Sero- A 57 cm long 10 mm diameter 30° forward oblique sanguinous fluid jetted out of the needle under pressure. viewing rigid laparoscope was introduced into the abdominal Analysis of the fluid revealed a total protein of 30 g/L, red blood cavity. An abnormally large volume of sero-sanguinous cell count of 0.39 9 1012 cells/L and total white blood cell count peritoneal fluid was visible within the peritoneal cavity of 0.69 9 109 cells/L. A packed cell volume value was not indicating either that the abdomen had not been drained provided by the analyser. significantly, or that the effusion had reformed in the interim 24 h. Insufflation of the abdomen with carbon dioxide was Treatment not performed as there was adequate visualisation of the Under sedation (romifidine 0.08 mg/kg bwt i.v., Sedivet2, plus ovarian pedicle. Only the dorsal aspect of the enlarged right butorphanol 0.01 mg/kg bwt i.v., Torbugesic4), the skin of the ovary could be visualised. The ovarian pedicle was injected ventro-cranial abdomen was prepared for aseptic surgery with 20 mLs 2% lignocaine (Lidocaine injection10) before and a 5 mm stab incision was made using a no. 11 blade being cauterised and transected using the LigasureTM Vessel through the skin, sub-cutis and linea alba before blunt Sealing System (Ligasure Atlas5). The pedicle was very thick introduction of a 10 cm long metal teat cannula through and the ligasure forceps were used to bluntly dissect it into the peritoneum in the midline. Approximately 30 L of lateral and medial sections prior to each cauterising bite, serosanguinous fluid were collected over 4 h. The horse’s because the full thickness of the pedicle was too large for heart rate was monitored during fluid collection to ensure it the forceps. Once the ovary was completely free of did not increase. Drainage was discontinued when the flow attachments within the abdomen, the laparoscopic trocars

were removed and portals closed in a routine fashion (Lloyd et al. 2007). General anaesthesia was then induced and the mare was positioned in dorsal recumbency. A 30 cm long ventral midline laparotomy incision was made at the level of the umbilicus and directed cranially to allow abdominal exploration. The right ovary was exteriorised via this incision and a rupture of the ovarian capsule was evident (Fig 5). Sectioning of the exteriorised ovary confirmed fresh haemorrhage and a haematoma adjacent to the area of the ruptured capsule (Fig 6). As there was still a copious volume of fluid within the abdomen, a 32F thoracic drain (standard trocar catheter11) was placed 10 cm lateral to the cranial aspect of the laparotomy incision with the finger of a surgical glove (with a slit at the distal end) sutured over its end to act as a one-way valve for egress of abdominal fluid. The laparotomy incision was closed routinely in three layers, and a stent was placed for recovery. Fig 6: Case 2: excised ovary sectioned, revealing fresh blood and haematoma formation adjacent to the capsular rupture Post-operative progress (arrow). The mare recovered uneventfully from surgery and the abdominal drain was removed 48 h post-operatively. She was discharged from the hospital 7 days after surgery. Follow-up 2004). Three cases of HP associated with haemorrhaging examination 6 weeks post-operatively revealed that the mare GCTs have also been reported by Dechant et al. (2006) was clinically well. Histological analysis of the excised ovary although more specific details of these cases were not given. confirmed a GCT. Although the risk of HP from GCTs appears to be low, early detection and surgical removal of GCTs is recommended to Discussion avoid this potentially life-threatening sequel. In women an increase in follicle-stimulating hormone (FSH) receptor Pathogenesis expression has been reported in the vascular endothelia on Active haemorrhage from GCT capsule rupture has been the periphery of tumours. The FSH binding these receptors in grossly confirmed as the cause of HP during laparotomy, turn is thought to promote neovascularisation (Radu et al. laparoscopy or post-mortem in horses (Gatewood et al. 1990; 2010). Increased FSH receptor expression resulting in Alexander et al. 2004; Harper et al. 2010; Sherlock et al. increased neovascularisation in the periphery of a tumour 2016). We suggest that rapid growth of the ovary may may be associated with severe haemorrhage caused by predispose to rupture of the ovarian capsule and capsular tears in larger GCTs in mares, although what haemorrhage however the cause is unclear. Both the GCTs in instigates the capsular tear is unknown. this report were large; however, GCTs of similar size have been described in the literature without associated bleeding Presentation and emergency stabilisation (Lloyd et al. 2007). In a previous case report, haemorrhaging This report highlights the fact that GCTs may occasionally GCTs weighing 4 and 8 kg were removed from mares with present with HP and, that in some cases, may require prompt HP, but size measurements were not noted (Alexander et al. medical resuscitation and emergency surgical intervention for a successful outcome, which, to the authors’ knowledge, has not previously been reported. Given the size of the tumours in this case report, it is likely that they had been present for much longer than the time period that clinical signs had been noted by the owners. These two cases presented with signs attributable to the HP i.e. anaemia and abdominal distension, and Case 1 also presented with collapse. This was in contrast to the typical behavioural signs that are more commonly present in mares with GCT, and which are attributable to the hormonal imbalances associated with these tumours. Identifying the cause of HP can sometimes be challenging, but in both cases in this report the presence of a very large, abnormal intra-abdominal mass was indicative of the instigating cause of haemorrhage. A previous case series described sub-acute intra- abdominal haemorrhage associated with GCTs in two mares (Alexander et al. 2004). Surgical treatment was performed electively following medical stabilisation for 10–21 days. Although this elective approach might be preferred, intra- Fig 5: Case 2: GCT capsule rupture; suspected to be the source abdominal haemorrhage can be rapid and severe in some of haemorrhage. cases, and can be successfully managed with prompt

surgical excision of bleeding GCTs. Acute emergency surgical Dechant et al. (2006) reported a survival rate of 44.7% at the intervention was undertaken in Case 1 which presented with time of discharge following medical management of HP severe anaemia and collapse because of the concern that however no specific treatment was significantly associated continued haemorrhage in an animal with an already low with survival. Supportive medical care may include i.v. fluid packed cell volume might result in fatal cardiovascular therapy, blood transfusions, nonsteroidal anti-inflammatories compromise. and antifibrinolytics (Dechant et al. 2006; Conwell et al. 2010; Stabilisation of hypovolaemic cases secondary to HP can Mudge 2014; Gray et al. 2015). include allogenic or autogenous blood transfusions, fluid Surgical removal of GCTs is the only solution available for therapy and antifibrinolytic agents such as aminocaproic treatment of the typical presentation of aggressive, stallion- acid. General guidelines for performing allogenic fresh whole like or nymphomania behaviour, infertility and anoestrous blood transfusions include typing both the donor and (McCue et al. 2006; Sherlock et al. 2016). Laparotomy recipient blood. Both major and minor cross matching is (Crabtree 2011) and laparoscopy have both been reported recommended (Hart 2011), but this is difficult in practice. for surgical removal of large, nonruptured GCTs (Rodgerson Fresh blood was collected separately from two donor horses et al. 2002; de Bont et al. 2010). Laparoscopic techniques are pre-surgery to be administered pre- and intraoperatively and currently favoured as they are minimally invasive and allow a further collection was taken and administered improved visualisation of the ovarian pedicle for ligation intraoperatively from a third donor horse. No cross matching (Hubert et al. 2006; Lloyd et al. 2007; de Bont et al. 2010), and was performed prior to the three fresh whole blood standing laparoscopic ovariectomy is associated with a lower transfusions as discussed by Durham (1996). Ideally cross post-operative complication rate versus conventional surgery matching should be performed for subsequent transfusions in mares (Lloyd et al. 2007; de Bont et al. 2010; Sherlock et al. after the first unmatched transfusion in an emergency (Hart 2016). Laparoscopic transection of the ovarian pedicle was 2011). Cross matching was performed post-operatively on performed in Case 2 24 h after abdominal drainage, however Case 1 to identify suitable donors for any subsequent a significant volume of fluid was still present in the abdomen allogenic transfusions if required. Although in an emergency which impeded visualisation to a degree. Repeat abdominal situation unmatched fresh whole blood may be used for the ultrasound examination following drainage and ideally on the first transfusion subsequent blood for transfusions should be day of surgery could have been performed in Case 2 to cross matched to prevent transfusion reactions (Durham gauge the remaining volume of HP. This would guide the 1996). The blood for transfusion was collected from hospital surgeon as to whether laparoscopic visualisation of the teaching mares as these were the only donors available; it is ovarian pedicle was likely to be adequate or not. However, if recommended that blood is taken from geldings to minimise the volume of HP is large or the case is unstable and therefore reactions occurring (Sellon and Wise 2010). Autotransfusion of not a suitable candidate for standing sedation (as in Case 1), abdominal blood collected from cases with HP has been laparoscopy is very unlikely to be successful and should steer described (Waguespack et al. 2001; Finding et al. 2011; the surgical choice towards laparotomy. Fouche et al. 2014). However, it is contraindicated if the HP is There are many choices for surgical ligation of thickened associated with neoplasia (Finding et al. 2011) or on-going ovarian pedicles including bipolar electrocautery with or haemorrhage (Hart 2011), and intraperitoneal blood is time without suturing, stapling of the mesovarium and the use of consuming to collect, therefore it may not be suitable for endoscopic clips (Doran et al. 1988; Lloyd et al. 2007; Smith unstable cases requiring prompt surgical treatment (Case 1). and Mair 2008; de Bont et al. 2010; Kummer et al. 2010; HP has been reported to have a high association with Goodin et al. 2011). Whichever method is used, care must be neoplastic causes (Dechant et al. 2006; Conwell et al. 2010) taken that haemostasis is complete and secure. and therefore, the sources of HP should be investigated Surgical removal of GCTs using a two-step procedure before autotransfusion is elected. (laparoscopic transection of the ovarian pedicle followed by In both cases a significant volume of HP was present, removal via ventral midline laparotomy) has been reported indicated by ultrasound findings. A subjective estimate of for removal of very large ovaries (up to 50 cm in size) (Vitte between 5 and 10 L was made for Case 1 (bwt 272 kg) et al. 2014), and this was the surgical method performed in following ultrasound examination and approximately 9 L of Case 2. Retrieval of very large tumours via a ventral midline blood was obtained at surgery, but it is likely that more blood laparotomy may be advantageous over making a very large was present in the depths of the abdomen. In Case 2 flank laparotomy incision in a standing case and lessened approximately 30 L of serosanguinous fluid was collected post-operative complications (Vitte et al. 2014). However, from the abdomen prior to laparoscopy. Drainage of the HP there is the additional associated risk and cost of general was only partially effective in Case 2 and unsuccessful in anaesthesia to consider. Alternatively, aspiration of the fluid Case 1, probably due to blood clots blocking the drainage contents of the mass or further dissection of enlarged ovaries cannula. If possible, controlled drainage is recommended with a morcellator into smaller size pieces in a specimen before general anaesthesia and exploratory laparotomy to retrieval bag via a flank incision has been reported (de Bont improve case stability and surgical visibility (Waguespack et al. 2010; Kummer et al. 2010), but we believe that this et al. 2001; Conwell et al. 2010; Finding et al. 2011). would not have been a realistic option in our two cases due to the size of the ovaries. Treatment options The reported prognosis for survival following medical Prognosis management of horses with HP is very variable (17–84%, There is a good prognosis following standing laparoscopic Pusterla et al. 2005; Dechant et al.2006; Arnold et al. 2008; removal of large, pathological ovaries and low complication Conwell et al. 2010). In cases taken to surgery the source of rates are reported: 99–100% of cases are reported to return to haemorrhage was identified in 77.7% (Conwell et al. 2010). their previous level of work and 85–93% of cases are

successfully bred within 30 months after surgery (Hubert et al. References 2006; Lloyd et al. 2007; de Bont et al. 2010; Kummer et al. 2010; € Alexander, G.R., Tweedie, M.A., Lescun, T.B. and McKinnon, A.O. Rocken et al. 2011; Kelmer et al. 2013). Short-term survival rates (2004) Haemoperitoneum secondary to granulosa cell tumour in for HP vary from 39 to 74% (Pusterla et al. 2005; Dechant et al. two mares. Aust. Vet. J. 82, 481-484. 2006; Conwell et al. 2010). Of the HP cases taken to surgery, a Arnold, C.E., Payne, M., Thompson, J.A., Slovis, N.M. and Bain, F.T. 42% survival rate has been reported (Conwell et al. 2010) and (2008) Periparturient haemorrhage in mares: 73 cases (1998-2005). surgical treatment of GCTs associated with HP was curative in J. Am. Vet. Assoc. 232, 1345-1351. all cases (Alexander et al. 2004; Dechant et al. 2006). de Bont, M.P., Wilderjans, H. and Simon, O. (2010) Standing laparoscopic ovariectomy technique with intrabdominal dissection for removal of large pathologic ovaries in mares. Vet. Surg. 39, Conclusion 373-741. Conwell, R.C., Hillyer, M.H., Mait, T.S., Pirie, R.S. and Clegg, P.D. (2010) In summary, GCTs should be considered as a differential Haemoperitoneum in horses: a retrospective review of 54 cases. diagnosis in mares presenting with HP. In some cases, the Vet. Rec. 167, 514-518. intra-abdominal haemorrhage is acute and severe and may Crabtree, J. (2011) Review of seven cases of granulosa cell tumour of necessitate emergency resuscitation of the case. Medical the equine ovary. Vet. Rec. 169, 251. stabilisation of the case is initially recommended and surgical Dechant, J.E., Nieto, J.F. and Le Jeune, S.S. (2006) Haemoperitoneum in removal of the affected ovary is likely to result in a successful horses: 67cases(1989-2004).J.Am.Vet.Med.Assoc. 229,253-258. outcome. Doran, R., Allen, D. and Gordon, B. (1988) Use ofstapling instruments to aid in the removal of ovarian tumours in mares. Equine Vet. J. 20, 37-40. Authors’ declaration of interests Durham, A.E. (1996) Blood and plasma transfusion in the horse. Equine Vet. Educ. 8, 8-12. fl No con icts of interest have been declared. Finding, E.J.T., Eliashar, E., Johns, I.C. and Dunkel, B. (2011) Autologous blood transfusion following an allogenic transfusion reaction in a case of acute anaemia due to intra-abdominal bleeding. Equine Ethical animal research Vet. Educ. 23, 339-342. This is a retrospective case report and as such no research Fouche, N., Cornelisse, K., Gerber, V., Gurtner, C., Krudewig, C. and animals were involved and ethical review was not Koch, C. (2014) Noncitrated blood transfusions used as adjunctive treatment in a 7-year-old Shetland Pony with haemoperitoneum applicable. Both cases were client owned, informed owner due to a ruptured corpus haemorrhagicum. Equine Vet. Educ. 26, consent was obtained for treatment and publication. Client 250-254. fi con dentiality has been maintained. Gatewood, D.M., Douglas, J.P., Cox, J.H., DeBowes, R.M. and Kennedy, G.A. (1990) Intra-abdominal haemorrhage associated with a granulosa-theca cell neoplasm in a mare. J. Am. Vet. Med. Source of funding Assoc. 196, 1827-1828. None. Goodin, J.T., Rodgerson, D.H. and Gomez, J.H. (2011) Standing hand- assistedlaparoscopicovariectomyin65mares.Vet.Surg.40,90-92. Gray, S.N., Dechant, J.E., LeJeune, S.S. and Nieto, J.E. (2015) Acknowledgements Identification, management and outcome of postoperative hemoperitoneum in 23 horses after emergency exploratory Thanks to J. Findley, E. Rioja-Garcia and all the staff members celiotomy for gastrointestinal disease. Vet. Surg. 44, 379-385. at the Philip Leverhulme Equine Hospital who were involved in Green, S.L., Specht, T.E., Dowling, S.C., Nixon, A.J., Wilson, J.H. and the diagnosis and treatment of Case 1. Thanks to J. Bryars Carrick, J.B. (1988) Hemoperitoneum caused by rupture of a and J. Garrett who were involved in the diagnosis of Case 2. juvenile granulosa cell tumor in an equine neonate. J. Am. Vet. Med. Assoc. 193, 1417-1419. Harper, J., Stewart, A.J., Kuhnt, L., Waguespack, R.W., Holland, M. and Authorship Downs, C. (2010) Ultrasonographic appearance and abdominal haemorrhage associated with a juvenile granulosa cell tumour in a F. Worsman assisted with management of Case 1 and wrote the foal. Equine Vet. Educ. 22, 115-120. majority of the manuscript. S. Barakzai managed Case 2, Hart, K.A. (2011) Pathogenesis, management and prevention of blood prepared Case 2 details for the manuscript and edited the transfusion reactions in horses. Equine Vet. Educ. 23, 343-345. manuscript. M. de Bont and L. Rubio-Martinez managed Case 1 Hubert, J.D., Burba, D.J. and Moore, R.M. (2006) Evaluation of a vessel- and edited the manuscript. S. Turner assisted with management sealing device for laparoscopic granulosa call tumour removal in of Case 2. All authors approved the final manuscript. standing mares. Vet. Surg. 35, 324-329. Kelmer, G., Raz, T., Berlin, D., Steinman, A. and Tatz, A.J. (2013) Standing open-flank approach for removal of enlarged pathologic Manufacturers' addresses ovaries in mares. Vet. Rec. 172, 687. 1 GE Medical Systems Limited, Chalfont St Giles, Buckinghamshire, UK. Kummer, M., Theiss, F., Jackson, M. and Furst,€ A. (2010) Evaluation of a 2 Boehringer Ingelheim Limited, Bracknell, Berkshire, UK. motorised morcellator for laparoscopic removal of granulosa- 3 Animalcare, York, UK. theca cell tumours in standing mares. Vet. Surg. 39, 649-653. 4Pfizer, Tadworth, Surrey, UK. Lloyd, D., Walmsley, J.P., Greet, T.R.C., Payne, R.J., Newton, J.R. and 5Covidien Surgical Solutions, Dublin, Ireland. Phillips, T.J. (2007) Electrosurgery as the sole means of haemostasis 6Fresenius Kabi, Runcorn, Cheshire, UK. during the laparoscopic removal of pathologically enlarged 7Dechra, Northwich, Cheshire, UK. ovaries in mares: a report of 55 cases. Equine Vet. J. 39, 210-214. 8Intervet, Milton Keynes, Buckinghamshire, UK. 9Norbrook, Corby, Northamptonshire, UK. McCue, P.M., Roser, J.F., Munro, C.J., Liu, I.K.M. and Lasley, B.L. (2006) 10Hameln Pharmaceuticals, Gloucester, UK. Granulosa cell tumours of the equine ovary. Vet. Clin. Equine Prac. 11Sims Portex Ltd, Hythe, Kent, UK. 22, 799-817.

Mudge, M.C. (2014) Acute haemorrhage and blood transfusions in Sherlock, C.E., Lott-Ellis, K., Bergren, A., Withers, J.M., Fews, D. and Mair, horses. Vet. Clin. North. Am. Equine Pract. 30, 427-436. T.S. (2016) Granulosa cell tumours in the mare: a review of 52 Pusterla, N., Fecteau, M.E., Madigan, J.E., Wilson, W.D. and cases. Equine Vet. Educ. 28, 75-82. Magdesian, K.G. (2005) Acute hemoperitoneum in horses: a review Smith, L.J. and Mair, T.S. (2008) Unilateral and bilateral laparoscopic of 19 cases (1992-2003). J. Vet. Intern. Med. 19, 344-347. ovariectomy of mares by electrcautery. Vet. Rec. 163, 297-300. Radu, A., Pichon, C., Camparo, P., Antoine, M., Allory, Y., Couvelard, Sundberg, J.P., Burnstein, T., Page, E.H., Kirkham, W.W. and Robinson, A., Fromont, G., Hai, M.T. and Ghinea, N. (2010) Expression of F.R. (1977) Neoplasms of equidae. J. Am. Vet. Med. Assoc. 170, follicle-stimulating hormone receptor in tumor blood vessels. N. 150-152. Engl. J. Med. 363, 1621-1630. Vitte, A., Rossignol, F., Mespoulhes-Riviere, C., Lechartier, A. and Rocken,€ M., Mosel, G., Seyrek-Intas, K., Seyrek-Intas, D., Litzke, F., Roecken,€ M. (2014) Two-step surgery combining standing Verver, J. and Rijkenhuizen, A.B.M. (2011) Unilateral and bilateral laparoscopy with recumbent ventral median celiotomy for laparoscopic ovariectomy in 157 mares: a retrospective removal of enlarged pathologic ovaries in 20 mares. Vet. Surg. 43, multicentre study. Vet. Surg. 40, 1009-1014. 663-667. Rodgerson, D.H., Brown, M.P., Watt, B.C., Keoughan, C.G. and Waguespack, R., Belknap, J. and Williams, A. (2001) Laparoscopic Hanrath, M. (2002) Hand-assisted laparoscopic technique for management of postcastration haemorrhage in a horse. Equine removal of ovarian tumours in standing mares. J. Am. Vet. Med. Vet. J. 33, 510-513. Assoc. 220, 1503-1507. White, R.A.S. and Allen, W.R. (1985) Use of ultrasound echography for Sellon, D.C and Wise, L.N. (2010) Disorders of the hematopoietic system. the differential diagnosis of a granulosa cell tumour in a mare. In: Equine Internal Medicine, 3rd edn. Eds: S.M. Reed, W.M. Bayly, Equine Vet. J. 17, 401-402. D.C. Sellon, Saunders, Elsevier, Inc., St. Louis, Missouri. pp 765-766.

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Case Report Septic pyomyositis, multiple pelvic osteomyelitis and thrombosis in a Thoroughbred foal M. C. Pressanto†* , F. Beccati†‡ , V. Stefanetti†, F. Passamonti†‡, N. Pilati† and M. Pepe†‡ †Department of Veterinary Medicine, Veterinary Teaching Hospital of the University of Perugia, Perugia; and ‡Sport Horse Study Center, Department of Veterinary Medicine, University of Perugia, Perugia, Italy. *Corresponding author email: [email protected]

Keywords: horse; foal; osteomyelitis; pyomyositis; arterial thrombosis; computed tomography

Summary a) b) A 1-month-old Thoroughbred colt was referred for acute hindlimb lameness. No history of illness or trauma was reported. The foal appeared responsive but reluctant to move. Clinical parameters revealed tachycardia (116 beats/min) and pyrexia (rectal temperature 39.8°C) with clear lung auscultation, normal appearance of the umbilicus, no diarrhoea and no appreciable joint swelling. The foal showed left hindlimb lameness (4/5 AAEP lameness c) d) scale), swelling of the croup and thigh and croup asymmetry. Complete blood count showed neutrophilic leucocytosis, increased serum amyloid A and fibrinogen concentrations. Ultrasound examination showed no significant abnormalities of the umbilicus or the abdomen. In the thorax, comet-tail artefacts were noted, bilaterally. Ultrasound examination of the croup and thigh revealed multiple circumscribed areas of echogenic fluid cranial to the left femur and in the caudal region of the left thigh, in addition to irregularity of the left acetabular and ischial tuberosity Fig 2: a) Left caudal gluteal/thigh region of the foal. Necrotic- bone surfaces. Medical treatment was started with broad- purulent flocculent material can be seen in the croup muscles. b) spectrum antibiotics and surgical treatment proposed. Acetabulum opened in transverse section. Areas of bone Radiographic and computed tomography (CT) examinations necrosis affected the physis and subchondral bone. c) Left of the pelvis were performed. Ventrodorsal and oblique projections internal iliac artery: septic embolus and wall thickening. The revealed extensive and irregular radiolucent areas surrounded by embolus extends approximately 8 cm inside the artery. d) sclerosis on the left acetabulum and osteolysis of the left ischial Pulmonary abscess found in the caudal lobe of the right lung. tuberosity and ischium. At CT scan, extensive areas of osseous hypoattenuation and severe irregularity were found affecting the left acetabulum (Fig 1), the left ischium bone and the left transverse A sterile sample collection of the lesions was performed and process of the sixth lumbar vertebra with involvement of the submitted for microbiological evaluation. After 24 h of intertransverse joint. At post-contrast venous phase, asymmetrical incubation, a pure growth of Streptococcus spp. was obtained. contrast enhancement was found on the left gluteal muscles. Due to the worsening of the clinical condition, the foal was subjected to euthanasia. At necropsy, left gluteal and caudal femoral muscles revealed pyomyositis (Fig 2a). The left acetabulum (Fig 2b) and sacral wing showed osteolytic areas. The lesions involving the left D ischial tuberosity and the left transverse process of L6 were confirmed. Evaluation of the ventral part of the sacroiliac area revealed an abnormal appearance of the left internal iliac

R L artery. Upon opening, a thrombus composed of purulent material was observed (Fig 2c). A nodular lesion was also found on the right lung (Fig 2d). Samples were collected from all lesions (lung, artery, muscle abscesses and bone) and a pure culture of S. zooepidemicus V was obtained from each sample. Fig 1: Transverse plane reformatted CT image of the acetabulum using maximal intensity projection (MIP). In the image, an extensive irregular hypoattenuating area (arrows) affecting the Key points ossiﬁcation centre of the acetabulum. • Computed tomography is a useful tool to diagnose involvement of multiple bones and soft tissue. • A S. zooepidemicus solitary lung abscess caused metastatic spread to different areas. • The Presence of a self-sustained local septic process was enhanced by an undetected septic thrombosis.

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Case Report Imaging ﬁndings associated with nasopharyngeal amyloidosis in an Arabian stallion L. A. Russell†, C. Navas de Solis†, C. M. Whitﬁeld-Cargile†, M. A. Deveau†, B. R. Buchanan‡ and L. J. Gilmour†* †Department of Large Animal Clinical Sciences, Texas A&M University, College Station, USA; and ‡Brazos Valley Equine Hospitals-Navasota, Texas, USA *Corresponding author email: [email protected]

Keywords: horse; computed tomography; magnetic resonance imaging; nasopharyngeal; amyloid

Summary 3 months during which the patient was hospitalised and A 3-year-old Arabian stallion presented for imaging and further boarded at the VMTH. Abdominal ultrasound, work-up of nasal discharge and respiratory stridor secondary to echocardiography, thoracic radiographs, urinalysis, and nasal amyloidosis. Thickening of the nasal mucosa with biopsies of the rectum and liver were normal. Abnormal heterogeneous attenuation of the affected tissues was identified findings on computed tomographic (CT), magnetic on CT images. The nasal mucosa, mucosa of the arytenoid resonance (MR), and endoscopic imaging are described cartilages, and regional lymph nodes were hypointense on T2 here. For CT, the patient was anaesthetised, positioned in and T1 MRI images with moderate, patchy contrast dorsal recumbency, and imaged with a 40 multislice helical 5 enhancement. Biopsy and histopathology of the nasal mucosa, scanner (Somatom Definition AS) using the following mucosa of the arytenoid cartilages and regional lymph nodes technical parameters: 120 kV, 233 mAs, 3 mm slices with an confirmed the original diagnosis of amyloidosis. Six fractions of interslice gap of 0.6 mm, tube rotation of 1.0 s, and pitch of helical tomotherapy did not provide significant improvement in 0.55. The field of view was set at 500 cm with matrix respiratory effort, and permanent tracheostomy was performed. dimensions of 512 9 512 cm to include the tip of the nose Haemorrhagic nasal discharge and mucosal plaques in the nasal rostrally through the atlanto-occipital joint caudally and cavity, pharynx, larynx, and trachea worsened over a 19-month encompassing the entire mediolateral width of the head. interim. This paper offers the first description of the CT and MRI Contiguous images were reconstructed in three standard findings of nasopharyngeal amyloidosis in a horse, and the first planes using bone and soft tissue algorithms. Window width description of the use of helical tomotherapy in an equine patient. and level were altered according to viewer preference. The CT showed mild thickening of the rostral mucosa of the left nasal passage. A focal region of the mucosa in the left nasal Signalment, history, and presenting complaint passage was rounded and heterogeneous in attenuation A 3-year-old Arabian stallion presented to the Veterinary (Fig 1a). Medical Teaching Hospital at Texas A&M University (VMTH) for The patient was positioned in right lateral recumbency for diagnostic imaging and evaluation of amyloidosis of the nasal MRI examination of the head to assess the severity of disease passages. The patient presented after clinical signs of respiratory progression and extent of tissues affected. The study was 5 stridor and nasal discharge did not respond to multimodal performed with a 3 Tesla system (Magnetom Verio) and a 6 therapy including courses of dexamethasone1 (0.05 mg/kg bwt transmit-receive 15-channel knee coil . Sequence parameters i.v. s.i.d. for 14 days, then per os s.i.d. for 14 days), flunixin were as follows: Precontrast 2D: turbo spin echo T2 weighted meglumine (Banamine2; 1.1 mg/kg bwt i.v. s.i.d. for 7 days), (T2W) transverse (TR 3500 ms, TE 82 ms, slice thickness 4 mm, pentoxyfylline3 (7.5 mg/kg bwt PF b.i.d. for 30 days), fluconazole1 interslice gap 0.4 mm, flip angle 132, FOV: 300, Matrix: (5 mg/kg bwt per os s.i.d. for 30 days), ceftiofur (Excede4; 512 9 432 cm), dorsal (TR 5290 ms, TE 89 ms, slice thickness 6.6 mg/kg bwt i.m., two treatments administered 4 days apart), 4 mm, interslice gap 0.4 mm, flip angle 131, FOV: 400, Matrix: and doxycycline1 (20 mg/kg bwt per os s.i.d. for 30 days). Biopsy 704 9 704 cm). Pre- and post-contrast 3D: T1 weighted of the nasal passages confirmed the presence of diffuse transverse, dorsal and sagittal (TR 2100, TE 3.89, TI 900 ms, slice plasmacytic rhinitis with abundant eosinophilic, amorphous thickness 0.78 mm, flip angle 12, FOV: 500, Matrix: material that was positive to Congo Red stain and consistent with 512 9 512 cm). Post-contrast sequences were acquired amyloid. On presentation to the VMTH, the patient was bright, 15 min following i.v. administration of 80 mL of 7 alert, and responsive with increased upper respiratory noise gadopentetate dimeglumine (Magnevist) . The MRI showed during inspiration and expiration. Serosanguinous nasal discharge thickening of the nasal mucosa, alar, and basal folds which was noted from the left nostril and there was decreased air almost completely occluded the right and left nasal flow from the left nostril when compared to the right. The passages. This thickened tissue contained regions of relative remainder of the physical examination was unremarkable. T2 and T1 hypointensity with moderate, patchy, and heterogeneous contrast enhancement of the nasal mucosa (Fig 1b–d). These changes were most severe at the rostral Imaging and diagnosis most extent of the nares, and the abnormal tissue extended To assess the patient for extent and severity of amyloidosis, a at least as far caudally as teeth 105/205 and 106/206 while variety of diagnostics were performed over a period of more normal-appearing tissue was noted caudal to teeth

a) c)

b) d)

Fig 1: Transverse plane images through the rostral nasal passage (a, b, c) and larynx (d) show thickened soft tissues (arrowheads), with the left side more affected than the right. The mucosa of the rostral nasal passage is rounded and heterogeneous in attenuation on CT (a), thickened with patchy hypointense regions on T2 weighted (b) and precontrast T1 weighted (c) MRI sequences. The mucosa of the arytenoid cartilages are enlarged, bulbous, and relatively hypointense on T2 weighted MR images (d), more severely on the patient’s left (arrowhead). The patient’s right side is on the left of each image.

108/208. The mandibular and medial retropharyngeal lymph along the left nasal septum with stippling and discoloration of nodes were enlarged bilaterally with mildly heterogeneous the nasal mucosa (Fig 2a and d). Subsequent endoscopy signal intensity on T2 weighted sequences. The mucosa of the procedures showed more severe reddening of the larynx and right and left arytenoid cartilages was asymmetrical, with pharynx with progressive enlargement of the mucosa of the small, bulbous areas of thickening along their ventral contour. right arytenoid cartilage (Fig 2b–c). The mucosa of the left arytenoid was larger than the mucosa Biopsies of the nasal mucosa, mucosa of the left of the right, and the bulbous regions were hypointense on T2 arytenoid cartilage, and submandibular lymph nodes were weighted images and hyperintense on T1 weighted images performed and confirmed amyloidosis of all tissues with when compared to the normal cartilage. Based on these additional evidence of granulomatous arytenoid chondritis, findings, amyloidosis of the nasal mucosa, mucosa of the mild lymphoid hyperplasia, and granulomatous arytenoid cartilages, and submandibular and medial lymphadenitis. Proteomic analysis showed the presence of retropharyngeal lymph nodes was suspected. apolipoprotein A-I, lambda light chain and no SAA protein Endoscopy of the upper airways was performed multiple suggesting localised AL (lambda) amyloidosis of the nasal times during the patient’s prolonged stay at the VMTH. and upper airway mucosa. Repeat examinations were indicated to evaluate progressively increasing respiratory effort and reduced nasal Treatment, outcome, and follow-up air flow. The first procedure revealed significant narrowing of the left nasal passage with enlargement of the soft tissues of Early in the course of work-up, the patient’s respiratory effort the medial aspect of the left nostril, nasal passage, and returned to normal following temporary tracheostomy. Due to

a) d)

b) e)

c) f)

Fig 2: Endoscopic images obtained at the time of initial work-up (a–d) compared with equivalent images obtained during follow-up assessment 15 months later (e–f). Progressive reddening, thickening, ulceration of nasal mucosa (a and d), pharyngeal mucosa (b and e), and mucosa of the arytenoid cartilages (c and f, arrowheads pointing to the left arytenoid), is noted over the recheck interim. The pharyngeal airway is almost entirely occluded by soft tissue thickening at the time of follow-up (e).

the multifocal distribution of amyloid identified via CT, MRI, improvement was appreciated following tomotherapy. A and endoscopy that was confirmed via histopathology, permanent tracheostomy was performed and the patient surgical removal was not attempted. Because of the lack of was discharged. options for medical management of amyloidosis in the horse, Communications with the owner 7 months after the last and based on reports of successful radiation therapy in the tomotherapy treatment revealed that the horse lived treatment of amyloidosis in humans, helical tomotherapy was comfortably as a breeding stallion. Mild haemorrhagic pursued. This consisted of a total radiation dose of 15.0 Gy to discharge from the nostrils and tracheostomy site was 95% of the planning target volume delivered in five fractions reported following exercise and rarely when semen was on an every other day schedule with weekends off. The gross collected. During this interim, nasal airflow was also tumour volume (GTV) was defined as gross identifiable subjectively improved when the tracheostomy site was disease on both CT and endoscopic evaluation. The GTV occluded. Fifteen months after initial presentation, the stallion margins were expanded 10 mm to encompass both was reassessed at the VMTH due to the increased microscopic extension (clinical target volume – CVT) and haemorrhagic discharge from the tracheostomy site and setup uncertainty (planning target volume – PTV). No clinical nostrils. The patient’s general physical examination was

normal with exception of red-brown staining of the hair Discussion around the tracheostomy site, submandibular lymph node enlargement, and visible plaques in the nasal passages. The Amyloidosis is a relatively rare disease in the equine patient horse was breathing normally though the permanent (Husby 1988; Van Andel et al. 1998) that refers to a number tracheostomy site. of grouped diseases occuring secondary to abnormal Complete blood count and serum chemistry were normal deposition of extracellular amyloid, an insoluble protein, with exception of mild hyperproteinemia (79 g/L, [reference within tissue, either systemically or locally. Both local and range, 53–73 g/L]) characterised by hyperglobulinaemia systemic forms of amyloidosis are recognised in humans and (44 g/L, [reference range 2.2–3.8 g/L]) and animals and may occur on their own, or in some cases, in hyperalbuminaemia (35 g/L, [reference range 23–31 g/L]). association with neoplasia (Shaw et al. 1987; Husby 1988; The mucosa of the nasal passages, pharynx, and larynx had Nappert et al. 1988; Mould et al. 1990; Oluk and Murphy large orange to yellow plaques that almost obliterated the 2010; Van Andel et al. 1998; Kim et al. 2005; Portela et al. airway, being much more extensive than those seen on 2012). The systemic form of amyloidosis in the horse is most previous endoscopic examinations (Fig 2e and f). In the nasal frequently the reactive or secondary form which is related to fl passages, the plaques were somewhat retracted into the chronic in ammatory disease and results in deposition of mucosa. Very small plaques were present in the floor of the amyloid apoprotein (AA type) (Kim et al. 2005; Gilad et al. medial compartment of the left and right guttural pouches. 2007). The primary form of amyloidosis (AL type) is rarely The tracheal mucosa was reddened with variably sized systemic and occurs secondary to abnormal amyloid plaques extending its entire length to the carina. A pool of production by plasma cells (Nappert et al. 1988; Kim et al. brown to red fluid was present in the thoracic inlet. 2005; Gilad et al. 2007). Amyloidosis can affect many Bronchoalveolar lavage fluid had a large amount of red different types of tissues, and the clinical signs reported are blood cells and haemosiderophages and was otherwise related to the tissue affected. In horses, AL amyloidosis cytologically normal. Ophthalmic examination showed frequently affects the nasal passages and upper respiratory haemolacria, and subconjunctival and conjunctival infiltrates tract and reported clinical signs include abnormal or increased in both eyes suspected to represent amyloid (Supplementary respiratory noise, exercise intolerance, upper airway Item 1); however, neoplastic or other infiltrates (such as obstruction secondary to thickening of the tissues, and squamous cell carcinoma or papilloma) could not be ruled epistaxis related to ulceration of the mucosa (Shaw et al. 1987; out. Thoracic radiographs were normal and unchanged Husby 1988; Nappert et al. 1988; Mould et al. 1990; Van Andel compared with those obtained 1 year prior. Ultrasound of the et al. 1998; Portela et al. 2012). Differential diagnoses include larynx and pharynx showed submandibular lymphadenopathy fungal rhinitis and sinusitis, such as secondary to Aspergillosis, and unremarkable appearance of the laryngeal cartilages. ethmoid haematoma, and neoplasia. Diagnosis may be fi Follow-up magnetic resonance imaging showed persistent con rmed via histopathology and affected tissues appear deposits consistent with the previously diagnosed amyloid with green under polarised light following staining with Congo Red. ’ fi focal reduction in soft tissue thickness of the nares and rostral To the authors knowledge, this is the rst report describing fi nasal mucosa compared with the prior study. While under the CT and MRI ndings of an equine patient with a fi anaesthesia for imaging, the horse developed hypotension con rmed diagnosis of nasal amyloidosis. The current fi that was poorly responsive to inotropes and the study was literature does not describe imaging abnormalities speci cto aborted before images of the larynx/pharynx could be amyloidosis, but abnormal areas noted on CT and MRI in the obtained. Biopsy samples of pharyngeal plaques showed present case correlate with affected tissues noted on histocytic and fibrosing pharyngitis without definitive evidence endoscopy and histopathology. CT and MRI have been used of amyloidosis on Congo red stain. in cases of amyloidosis in humans to monitor progression of fi While not confirmed histopathologically, worsening disease but speci c changes associated with the disease are amyloidosis remained high on the differential list for not reported (Gilad et al. 2007; Oluk and Murphy 2010). In progressive plaques in the mucosa of the larynx, pharynx, human cases of amyloidosis, T2W hypointense lesions with trachea, and periocular tissues. Re-irradiation of this patient variable contrast enhancement are described, similar to was not pursued for a number of reasons, driven principally those seen in this patient (Marton et al. 1991; Panda et al. by the clinically disappointing response to the first course of 2007). The most commonly described treatment in horses is fi radiation therapy and the perception of increased con ned to surgical excision, and the success of this is anaesthetic risk during repeated anaesthetic procedures. dependent on the degree of dissemination of disease. Extension of amyloid deposits beyond the larynx precluded Unfortunately, based on the imaging and corroborating fi treatment of all affected areas. Since helical tomotherapy histological ndings, the extent of the disease precluded and CT share similar designs, longitudinal treatment surgical excision in this patient. Focal radiation, as a sole distance is limited by how much volume of the patient will therapy or in conjunction with surgery, is reported in humans fit into the bore. In this patient, the disease had progressed to decrease the size of masses, arrest the progression of far beyond the original treatment field, and beyond the disease and improve the symptoms associated with volume limitations of helical tomotherapy. Referral to amyloidosis (Monroe et al. 2004; Neuner et al. 2010; Oluk and ’ another institution with a conventional linear accelerator to Murphy 2010; Truong et al. 2011). To the authors knowledge, fi retreat the previous site as well as new areas of progression this is also the rst report to describe the use of helical deeper into the head and neck was declined by the tomotherapy in veterinary medicine on an equine patient as fi owner. well as being the rst report to describe its use for treatment ’ The patient was euthanised 22 months following initial of amyloidosis. Unfortunately, in this case, the patient s fi presentation due to progressively worsening clinical signs and clinical signs did not signi cantly improve despite treatment. It deteriorating quality of life perceived by the owner. is uncertain if a different treatment regimen or combination

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with surgical debulking could have led to outcomes more 2Merck Animal Health, Madison, New Jersey, USA. 3 similar to those reported in human medicine. The radiation Apotex Corporation, Toronto, Ontaria, Canada. 4Zoetis, Parsippany, New Jersey, USA. protocol used in this case varied from those reported in the 5Siemens, Malvern, Pennsylvania, USA. human literature. The principal reason for this change is the 6Quality Electrodynamics, Cleveland, Ohio, USA. constraint of requiring general anaesthesia for radiation 7Bayer Healthcare Pharmaceuticals, Inc., Wayne, New Jersey, USA. therapy in veterinary patients. Due to this constraint, a fi decreased number of fractions ( ve instead of 10) was References prescribed, and due to the young age of the patient and concern for radiation therapy related toxicities, the dose per Gilad, R., Milillo, P. and Som, P.M. (2007) Severe diffuse systemic fraction was not increased. Including the entire protocol for amyloidosis with involvement of the pharynx, larynx, and trachea: CT and MR findings. Am. J. Neuroradiol. 28, 1557-1558. the radiation treatment of this patient and comparing it to Husby, G. (1988) Equine amyloidosis. Equine Vet. J. 20, 235-238. those used in human medicine is considered beyond the scope of this paper. Histopathology correlated with MRI Kim, D.Y., Taylor, H.W., Eades, S.C. and Cho, D.Y. (2005) Systemic AL fi amyloid associated with multiple myeloma in a horse. Vet. Pathol. ndings that helped characterize the extent of locoregional 42, 81-84. disease involving the nasal passages, arytenoids, and lymph Marton, A.D., Clandia, F.E. and Kirsch, B.S. (1991) Focal amyloidosis of nodes. This case highlights the use of multimodal imaging in the head and neck: evaluation with CT and MR Imaging. the work-up and diagnosis of amyloidosis in a horse, with Radiology 181, 521-525. palliative treatment able to prolong good quality of life and Monroe, A.T., Walia, R., Zlotecki, R.A. and Jantz, M.A. (2004) function as a breeding stallion. Inability to arrest the long term Tracheobronchial amyloidosis: a case report of successful progression of this disease and/or its symptoms despite many treatment with external beam radiation therapy. Chest 125, 784- therapeutic attempts underscores the need for more 789. effective therapeutic options for amyloidosis. Mould, J.R., Munroe, G.A., Eckersall, P.D., Conner, J.G. and McNeil, P.E. (1990) Conjunctival and nasal amyloidosis in a horse. Equine Vet. J. 22, 8-11. Authors’ declaration of interests Nappert, G., Vrins, A., Dore, M., Morin, M. and Beauregard, M. (1988) Nasal amyloidosis in two quarter horses. Can. Vet. J. 29, 834-835. No conflicts of interests have been declared. Neuner, G.A., Badros, A.A., Meyer, T.K., Nanaji, N.M. and Regine, W.F. (2010) Complete resolution of laryngeal amyloidosis with radiation Ethical animal research treatment. Head Neck 34, 748-752. Oluk, M.A. and Murphy, J. (2010) Nasopharyngeal amyloidosis: an This case report describes the presentation, diagnostic work- unusual cause for epistaxis. J. Laryngol. Otol. 124, 209-212. up, diagnosis and therapy of a single horse that was treated Panda, N.K., Saravanan, K., Purushotaman, G.P., Gurunathan, R.K. to a high standard of veterinary care by both the referring and Mahesha, V. (2007) Localized amyloidosis masquerading as veterinarian and the submitting institution. Client consent was a nasopharyngeal tumor: a review. Am. J. Otolaryngol. 28, 208- obtained prior to manuscript preparation, and client 211. confidentiality is maintained on the submitted figures. Portela, R.A., Dantas, A.F., de Melo, D.B., Marinho, J.M., Neto, P.I. and Riet-Correa, F. (2012) Nasal amyloidosis in a horse. Braz. J. Vet. Pathol. 5, 86-88. Source of funding Shaw, D.P., Gunson, D.E. and Evans, L.H. (1987) Nasal amyloidosis in four horses. Vet. Pathol. 24, 183-185. None. Truong, M.T., Kachnic, L.A., Grillone, G.A., Bohrs, H.K., Lee, R., Sakai, O. and Berk, J.L. (2011) Long-term results of conformal radiotherapy Acknowledgements for progressive airway amyloidosis. Int. J. Radiat. Oncol. Biol. Phys. 83, 734-739. The authors would like to thank Dr Julian Gillmore and Dr Van Andel, A.C., Gruys, E. and Kroneman, J. (1998) Amyloid in the Rodney H. Falk for assistance in interpretation of biopsy horse: a report of nine cases. Equine Vet. J. 20, 227-285. samples, Douglas R. Cummings for assistance in editing images for publication, and Denise Shannon for patient Supporting information monitoring after hospital discharge. Additional Supporting Information may be found in the online Authorship version of this article at the publisher’s website: All authors contributed equally in preparation, editing and Supplementary Item 1: Fifteen months following initial final approval of this manuscript. This case report did not evaluation at the VMTH, ophthalmic examination revealed require formal study design, execution or data analysis. subconjunctival haemorrhage (asterisk), and subconjunctival and conjunctival infiltrates in both eyes (arrows). This material was suspected to represent amyloid deposition; however, Manufacturers' addresses biopsies were not pursued and neoplastic or inflammatory 1Professional Compounding Centers of America, Houston, Texas, USA. infiltrates remained on the differential list.

Clinical Commentary Equine external beam radiation therapy: Techniques and challenges N. C. Desai* and E. M. Green Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA *Corresponding author email: [email protected]

Keywords: horse; radiation therapy; external beam radiation therapy; radiotherapy; linear accelerator

Summary Radiation therapy is becoming increasingly more available and in demand for small animal veterinary cases. Although there is a demand for radiation therapy in large animal cases, there are certain limitations of external beam radiation therapy for horses that restrict treatment options. Currently, there are only ﬁve veterinary teaching hospitals in the United Gantry States where external beam radiation therapy can be performed on horses and other large animals. This is in contrast to the roughly 80 facilities, both academic and private, available for small animals. The goal of this commentary is to give a brief overview of external beam radiation therapy and highlight the steps involved, and the technical difﬁculties faced, while treating horses with external beam radiation therapy.

Methods of radiation delivery

There are a number of ways radiation can be delivered: Couch external beam radiation therapy, plesiotherapy, and interstitial brachytherapy. External beam radiation is the most commonly used and available modality in veterinary medicine. External beam radiation therapy uses a machine to aim x-ray or gamma ray photons or electrons at a tumour Flat panel from a distance. X-ray photons and electrons are delivered from linear accelerators (Fig 1a,b); helical tomotherapy units (Fig 2) deliver x-ray photons; and Co60 machines deliver gamma ray photons. Most veterinary facilities use linear accelerators with photon energies ranging from 4 to 8 MV (Farrelly and McEntee 2014) and electron energies from 6 to 21 MeV., Energy of the photon or electron dictates its depth a) of penetration. Plesiotherapy is the application of beta (electron) radiation therapy to the surface of a tumour using a radioactive source such as Strontium (Sr90). It is typically used for small, superﬁcial lesions like ocular squamous cell Gantry carcinoma (Plummer et al. 2007) and dermal lesions as the depth of treatment is roughly 3 mm (Menon and Sloboda 2000). Interstitial brachytherapy involves the implantation of

Flat panel Fig 1: a) and b) An ONCORTM Digital Medical Linear Accelerator with on board imaging, IMRT and IGRT capability. The gantry can rotate through 360° around the patient couch allowing the targeting of the tumour from different angles. Onboard imaging assures accurate positioning of the cases prior to treatment delivery. For onboard imaging, the digital imaging flat panel is extended and an image is made using an MV beam. Once imaging is performed and accurate patient positioning is verified, the flat panel is retracted for treatment. b) Note the rotation of the b) gantry.

radioactive sources emitting gamma ray photons or electrons increased (dose escalation) to achieve greater tumour (typically iridium-192 or cesium-131 or 137) within, or near, the control. tumour (Ramachandran 2017). All these methods are used to treat horses with radiation therapy but this commentary will Conformal treatment techniques focus on external beam techniques. Conformal radiation therapy techniques use cross-sectional External beam radiation therapy images (CT and MRI) and computerised treatment planning to design radiation plans that target tumours with multiple There are different techniques by which external beam beams (coplanar and non-coplanar) delivered from different radiation can be delivered. Electrons are used to treat angles. 3DCRT plans uses multiple coplanar beams to target superficial lesion like sarcoids and other cutaneous tumours the tumour volume. The fields are shaped to the tumour and scars (McDermott and Orton 2010a), whereas photons volume using a multileaf collimator (MLC) (Fig 3). An MLC is a are used to treat deeper tumours (Khan and Gibbons 2014a) device consisting of 0.5 to 1.0 cm thick ‘leaves’ of a high (e.g. lacrimal duct tumours, nasal and oral tumours). The atomic number material (typically tungsten) that are location of the tumour and the radiation sensitivity of individually movable and are used to block portions of the adjacent normal tissues is taken into consideration when beam (McDermott and Orton 2010b), IMRT differs from 3DCRT determining beam orientation. For tumours not adjacent to by delivering multiple fields (often up to 15) from each beam sensitive normal tissues, a single beam or parallel-opposed angle. The MLC is used to shape each field, often to areas beams may be appropriate and the calculation of the smaller than the tumour volume, and by doing so, modulates machine parameters can be performed by hand (i.e. the intensity of the radiation beam across the treated manual calculations). With such a plan, avoiding radiation volume. This dose modulation enhances dose conformity to dose to normal tissues adjacent the tumour is difficult and the the tumour and more effectively reduces radiation dose to entire treated field (tumour and normal tissues) receives the normal tissues (Khan and Gibbons 2014b). SRT plans uses same dose. multiple (often non-coplanar) tightly collimated beams to The significant developments in the field of veterinary deliver very high doses of radiation in a single or a few (3–5) imaging using CT and MRI to image tumours translate to treatments (Tannock et al. 2013). Co60 machines can be advances in radiation oncology. Accurate delineation of used for 3DCRT plans while linear accelerators equipped with tumour margins using cross-sectional imaging allows for more MLCs can deliver 3DCRT, IMRT and SRT plans. Helical advanced conformal treatment techniques like 3D conformal TomoTherapyTM machines are used to deliver IMRT and SRT radiation therapy (3DCRT), intensity modulated radiation plans. therapy (IMRT), image guided radiation therapy (IGRT) and As highly conformal radiation delivery techniques are now stereotactic radiation therapy (SRT) to deliver radiation plans. frequently used in veterinary radiation oncology, there is a Using these new techniques, veterinary radiation oncologists greater need to ensure accurate, repeatable positioning of can now target the tumour more precisely and limit the dose the cases. Without precise positioning, the normal tissue to normal tissues. Radiation dose to the tumour can then be sparing advantage of conformal techniques is lost. Appropriate positioning of the cases is achieved using immobilisation devices like bite blocks and Vac-LokTM mattresses (Fig 4a,b).

Fig 3: Digitally reconstructed radiograph of a horse treated for a nasolacrimal duct carcinoma delineating the radiation treatment ﬁeld (yellow box). Each MLC ‘leaf’ is outlined in blue and the Fig 2: TomoTherapyTM is a linear accelerator that sits in a CT tumour is outlined in orange. Note how the MLC positions shape gantry. Radiation is delivered slice by slice as the patient is the radiation ﬁeld to the margins of the tumour, allowing the advanced, helically combining IMRT and IGRT. TomoTherapyTM collimation of the radiation beam and modulation of the intensity does not have electron capabilities. of radiation within the tumour.

Vac-LokTM Treated area mattress

Bite block & mouth mould

b) b) Fig 4: a) Image of a dog placed in a bite block (acrylic), dental mouth mould (dark orange material), and Vac-LokTM mattress Fig 5: a) Portal image, 2-dimensional image of a 6-year-old dog minimising daily set up error. b) Image of horse in a Vac-LokTM treated for a plasma cell tumour of the tarsus. Note the treatment fi mattress. eld in the centre (white area). b) Image guided radiation therapy: MVCT. 3-dimensional imaging. The portion of the image shaded blue is the stimulation CT (KVCT) scan performed for An immobilisation device is used to position a patient radiation therapy planning. The portion of the image without for a radiation planning CT scan. The same immobilisation shading is the MVCT scan performed prior to treatment delivery fi device is then used for patient positioning during each for position veri cation. The two images are superimposed and the MVCT portion adjusted to ensure accurate patient positioning. radiation treatment. Furthermore, to ensure accurate The tumour volume is outlined in orange and blue and the brain positioning, image guidance is required. Image guided in pink. radiation therapy (IGRT) uses either 2-dimentional (double exposure portal images) or 3-dimensional (cone beam CT) images to verify the patient’s position prior to dose delivery. particularly true when using advanced conformal radiation IGRT is a technique used in combination with 3DCRT, IMRT therapy techniques. For superficial tumours like tumours and SRT radiation treatments. Linear accelerators involving the skin, this ‘tumour delineation’ can be done by equipped with on-board digital imaging panels can make measuring the skin lesion. When deeper tissues are involved, portal images and either cone beam CT images while further imaging using a radiograph (for bone tumours), a CT TomoTherapyTM units can only make helical KVCT scan, or an MRI is required to accurately define the tumour images (Fig 5a,b). Portal images are typically used to verify volume. After delineating the extent of the tumour, normal patient positioning for simple, non-conformal plans. Cone critical tissues are identified. If a computerised plan is beam CT images are necessary for conformal plans using required for conformal treatment techniques, the CT or MRI IMRT or SRT. images are loaded into a planning computer and the tumour and normal critical tissues are contoured (Fig 6). The radiation Steps involved in external beam radiation oncologist determines the number and orientation of beams planning and then the computer calculates the dose to all the contoured structures. This information is displayed as a dose For optimal tumour control and outcome, precise delineation volume histogram (DVH), a graphical representation of the of the tumour volume is the most important step. This is dose in given volumes of the tumour and normal structures

(Fig 7). If the conformal plan is deemed acceptable, a Left eye physicist performs quality assurance to make sure the dose delivered by the machine matches that of the computer plan prior to treating the patient.

Tumour Technical difﬁculties associated with external beam radiation in horses The advanced conformal techniques described above are frequently used for external beam radiation in small animals; however, there are certain limitations when treating horses. Advanced imaging modalities such as CT and MRI, necessary to plan the conformal techniques, are challenging in horses for several reasons. First, to perform these studies, a special table, designed to accommodate the horse’s weight, is needed. These tables must be made of MRI compatible metals for MRI use and those used for CT must interface with Fig 6: Contouring: A CT scan of a cat with a nasal tumour the CT couch to control the patient’s movement. Such tables demonstrating contouring. The tumour is outlined in orange and are commercially manufactured and available at several the normal structures in green (right eye), light green (right lens), blue (left eye) and light blue (left lens). private practices and universities. Second, these tables do not

Fig 7: After appropriate beam angle placement and computerised planning a dose volume histogram is generated (DVH) which shows the dose to normal tissues and the tumour. Each colour corresponds to a normal tissue or the tumour. Tissue volumes are depicted on the Y axis and the X axis represents the radiation dose received. DVHs allow evaluation of a treatment plan to determine if the tumour (orange, red and blue lines towards the right) is receiving the prescribed dose (42 Gy) and the doses to the normal structures (lines towards the left) are acceptable.

fit into the MRI bore or CT gantry so only limited anatomy can be imaged. Tumours in the upper limbs, caudal neck, thorax, abdomen and pelvis cannot be imaged by these modalities, making advanced conformal radiation therapy options impossible in these areas. Another obstacle to treating horses with external beam radiation therapy is access to the radiation machine. Radiation therapy suites are typically designed with standard entry door and a hallway with a 90-degree angle (referred to as the maze) leading into the main room that prevents rolling a horse on a table into the suite. Only five veterinary teaching hospitals have larger doors and a means to Surgery table negotiate or bypass the maze to accommodate horses in couch their radiation suites. Once the horse is in the room, the radiation therapy couch has to be completely moved out of the way to make room for the table the horse is on (Fig 8a, a) b). Due to its size, the horse table also limits certain gantry angles making 3DCRT or IMRT plans challenging. The gantry will not fit under the table and angles from 110-degrees to 250-degrees cannot be used in treatment plans. This is important to keep in mind when the radiation planning cross- sectional imaging is performed. As most horses are placed in dorsal recumbency for imaging and treatment as they are under general anaesthesia, tumours in the dorsal aspect of the head may be difficult to treat with the restricted gantry angle unless the head is rotated. It is also possible that portal imaging and cone beam CTs needed to confirm appropriate patient positioning cannot be performed due to the inability to access the flat panel and restricted gantry positions. Due to the limitations listed above, considerable forethought is necessary prior to any equine radiation therapy plan.

b) Conclusion Fig 8: a) and b) A horse treated for a nasolacrimal duct In conclusion, advanced conformal radiation techniques are carcinoma. The linear accelerator couch is moved out of the now frequently used in veterinary radiation oncology. These way and an equine surgery table with the horse are moved in are still mainly restricted to small animal cases due to speciﬁc position for treatment. Note that the table prevents the gantry technical challenges; however, advances are being made in from moving under the horse. Beam angles between 110° and ﬁ 250° cannot be used. the eld of equine radiation oncology to use these techniques to treat horses.

Authors’ declaration of interests No conﬂicts of interest have been declared.

Ethical animal research Owner consent has been taken to use the images for educational purposes.

Source of funding None.

Authorship N. Desai was responsible for the preparation and editing of Fig 9: A horse being treated for a recurrent limb haemangioma the manuscript and E. Green was responsible for editing the ﬁ with parallel-opposed ﬁelds. Note the couch is pushed out of the manuscript. Both authors gave their nal approval for the way to make place for the equine surgery table. manuscript.

References McDermott, Medical Physics Publishing, Madison, Wisconsin. pp 13-16. Farrelly, J. and McEntee, M.C. (2014) A survey of veterinary radiation Menon, G. and Sloboda, R. (2000) Measurement of relative output for facilities in 2010. Vet. Radiol. Ultrasound. 55, 638-643. https://doi. 90Sr ophthalmic applicators using radiochromic ﬁlm. Med. Dosim. org/10.1111/vru.12161. 25, 171-177. Khan, F.M. and Gibbons, J.P. (2014a) Dose distribution and scatter Plummer, C.E., Smith, S., Andrew, S.E., Lassaline, M.E., Gelatt, K.N., analysis. In: The Physics of Radiation Therapy, 5th edn., Ed: J.W. Brooks, D.E. and Ollivier, F.J. (2007) Combined keratectomy, Pine, Jr., Lippincott Williams & Wilkins, Philadelphia. pp 137. strontium-90 irradiation and permanent bulbar conjunctival grafts Khan, F.M. and Gibbons, J.P. (2014b) Intensity modulated radiation for corneolimbal squamous cell carcinomas in horses (1990-2002): therapy. In: The Physics of Radiation Therapy, 5th edn., Ed: J.W. 38 Horses. Vet. Ophthalmol. 10, 37-42. https://doi.org/10.1111/j. Pine, Jr., Lippincott Williams & Wilkins, Philadelphia. pp 430. 1463-5224.2007.00489.x. McDermott, P.N. and Orton, C.G. (2010a) Electron beam radiation Ramachandran, P. (2017) New era of electronic brachytherapy. World therapy. The Physics and Technology of Radiation Therapy, 1st edn., J. Radiol. 9, 148-154. https://doi.org/10.4329/wjr.v9.i4.148. Ed: P.N. McDermott, Medical Physics Publishing, Madison Wisconsin. Tannock, I., Hill, R., Bristow, R. and Orton, C.G. (2013) Tumor and pp 15-1. normal tissue response to radiation therapy. The Basic Science of McDermott, P.N. and Orton, C.G. (2010b) Shaped Fields. The Physics Oncology, 5th edn., Eds: I.F. Tannock, R.G. Birstow, R.P. Hill, L. and Technology of Radiation Therapy, 1st edn., Ed: P.N. Harrington. McGraw- Hill Education, China. pp 358.

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Original Article Atypical myopathy in the South-East of England: Clinicopathological data and outcome in hospitalised horses B. Dunkel†* , A. Ryan†, E. Haggett‡ and E. J. Knowles§ †Department of Clinical Science and Services, The Royal Veterinary College, North Mymms, Hertfordshire; ‡Rossdales Equine Hospital and Diagnostic Centre, Newmarket, Suffolk; and §Bell Equine Veterinary Clinic, Mereworth, Maidstone, Kent, UK *Corresponding author email: [email protected]

Keywords: horse; myopathy; myoglobinuria; sycamore; lactate; triglycerides

Summary odds of survival were recorded in certain geographic This retrospective case series describes clinicopathological locations (Gonzalez-Medina et al. 2017). To date, no study data and outcome of hospitalised atypical myopathy has reported survival exclusively in hospitalised horses or (AM) cases in the South-East of England. The study aimed to specifically the South-East of England, where the disease is describe the frequency of metabolic abnormalities particularly common (Gonzalez-Medina et al. 2017). (hyperglycaemia, hyperlactataemia, hypertriglyceridaemia) Nonsurvivors mostly die or are subjected to euthanasia within and outcome in AM cases in the South-East of England and 72 h of onset of clinical signs (Hosie et al. 1986; Whitwell et al. test the hypothesis that serum creatine kinase (CK) activity 1988; Votion et al. 2007, 2014; Westermann et al. 2008; van and blood glucose, lactate and triglyceride concentrations Galen et al. 2012a). Currently, ingestion of the toxin are associated with outcome. Medical records (2011–2017) hypoglycin A is thought to be responsible for the observed from three referral hospitals were reviewed for cases with a clinicopathological changes (van Galen et al. 2012b; Votion clinical diagnosis of AM. A previously described algorithm et al. 2014). Hypoglycin A causes an acquired multiple was applied and cases were included if a diagnosis of AM acyl-CoA dehydrogenase deficiency, impairing mitochondrial was considered highly likely. In cases admitted after 2013 metabolism and leading to excessive lipid accumulation in known or possible exposure to sycamore trees was also myofibres (Westermann et al. 2008; Sponseller et al. 2012). required for inclusion. Sixty-four animals were included, 44% Hypoglycin A is metabolised into methylenecyclopropyl (28/64) survived. Hyperglycaemia, hyperlactataemia and acetic acid-CoA (MCPA-CoA), which binds irreversibly to hypertriglyceridaemia were present in 76%, 89% and 92% of acyl-CoA. This inhibits several acyl-CoA dehydrogenases horses on admission, respectively. Survivors had lower blood (Meda et al. 1999; Joskow et al. 2006) which in turn block a lactate concentrations (survivors: median 3.5 mmol/L; range number of steps in the mitochondrial lipid metabolism, causing 0.5–10.4 mmol/L vs. nonsurvivors: median 7.3 mmol/L; range an energy deficiency. Clinical signs of AM are caused by 2.5–16.5 mmol/L; P = 0.011) and serum CK activities (survivors: severe rhabdomyolysis of type 1 myofibres and include acute median 38,369 U/L; range 7024–570,498 U/L vs. nonsurvivors: stiffness, muscle fasciculation, sweating, respiratory difficulty median 172,687 U/L; range 2036–570,953 U/L; P = 0.027) on and weakness, often progressing to recumbency and death admission when compared to nonsurvivors. Increasing CK (Cassart et al. 2007; Palencia and Rivero 2007; Votion et al. activity (P = 0.008) and triglyceride concentrations (P = 0.038) 2007). Increased serum activity of creatine kinase (CK), during hospitalisation were associated with nonsurvival. More aspartate amino transferase (AST) and lactate dehydrogenase nonsurvivors required sedation (18/29; 62.1% vs. 4/22; 18.2%; as a consequence of severe rhabdomyolysis is frequently P = 0.002). The prognosis for hospitalised horses with AM is observed in AM-affected horses. Although in earlier studies, the guarded and outcome in this population was associated with magnitude of enzyme activity increases has not been admission CK activity and lactate concentrations, and associated with outcome, two recent studies did identify an increasing CK activity and triglyceride concentrations and association between CK activities and survival (Boemer et al. need for sedation during hospitalisation. 2017; Gonzalez-Medina et al. 2017). In addition, abnormalities of energy metabolism including hyperglycaemia, hypertriglyceridaemia and hyperlactataemia have been Introduction reported in earlier and one recent report (Finno et al. 2006; Westermann et al. 2008; Boemer et al. 2017) but information Atypical myopathy (AM) and seasonal pasture myopathy in on the frequency and magnitude of these metabolic the USA are seasonal, acute myopathies of grazing horses derangements in clinical patients and their association with (Whitwell et al. 1988; Finno et al. 2006; Votion et al. 2009; van outcome remains sparse. In recent years, AM has received Galen et al. 2012a) and both are believed to have similar increasing attention in the UK and anecdotally based on aetiologies (van Galen et al. 2012b; Sponseller et al. 2012; subjective impression, it appears that survival of hospitalised Westermann et al. 2016). A high mortality rate ranging cases has improved over time. Although a recent study did between 74 and 97% has been reported in European not identify differences in glucose, lactate and triglyceride outbreaks while a recent study documented a slightly lower concentrations between survivors and nonsurvivors (Boemer mortality of 61% in UK cases (Gonzalez-Medina et al. 2017). et al. 2017), clinically, there seems to be a possible Within the UK, differences in numbers of cases and in the correlation in hospitalised horses.

The aims and objectives of this study were therefore to triglyceride concentrations on admission) were analysed determine the frequency and extent of metabolic using a student’s t test (normally distributed data) or abnormalities, particularly hyperglycaemia, hyperlactataemia Mann–Whitney U test (non-normally distributed data). The and hypertriglyceridaemia and to establish the outcome and correlation between glucose and lactate concentrations and clinicopathological findings associated with outcome in glucose and triglyceride concentrations on admission were hospitalised horses suffering with AM in three referral hospitals investigated using a Spearman’s correlation. Statistical in the South-East of England. The study tested the hypothesis significance was set at P≤0.05. that admission serum CK activity and glucose, lactate and fi triglyceride concentrations were signi cantly associated with Results outcome in hospitalised horses with AM. Sixty-four horses met the inclusion criteria, 15 from the RVC, Materials and methods 25 from BEVC and 24 from REHDC. The median age of 25 mares, 35 geldings and four stallions was 5 years (range Medical records from January 2011 to August 2017 from three 0.25–22 years); age and sex were not associated with equine referral hospitals in the South-East of England (the outcome (P = 0.32 and P = 0.5, respectively). Breeds were 20 Royal Veterinary College Equine Referral Hospital [RVC], Bell Cobs and Cob crosses, 13 Thoroughbreds and Thoroughbred Equine Veterinary Clinic [BEVC] and Rossdales Equine Hospital crosses, nine Irish draughts and crosses, seven Warmbloods, and Diagnostic Centre [REHDC]) were searched for horses four Connemara ponies, three Welsh ponies, two Arabians with a diagnosis of AM. A diagnosis was established based on and one Andalusian, New Forest pony, native pony and clinical (clinical signs and biochemistry changes compatible Miniature Shetland each. In two horses, a breed was not with a myopathy) or post-mortem findings in a horse kept at recorded. The date of presentation was recorded for 27 pasture by following a diagnostic algorithm for AM that has cases: seven (26%) presented in winter (December–January), been previously described and used in other studies (van eight (30%) in spring (March–May) and 12 (44%) in autumn Galen et al. 2012a; Boemer et al. 2017; Gonzalez-Medina (September–November). et al. 2017). In cases admitted after Acer pseudoplantanus Hyperglycaemia (reference range 4.2–6.7 mmol/L) was (sycamore trees) had been identified as source of hypoglycin present in 28/37 (76%) of cases on admission, while one horse A, known or an possible exposure to sycamore trees was also presented with hypoglycaemia. Plasma lactate (reference included as an essential criterion. Horses with a known history range 0.2–2.5 mmol/L) and triglyceride concentrations of exertional myopathy or of rigorous exercise prior to (reference range 0.17–0.46 mmol/L) were increased in 31/35 development of clinical signs were excluded. In addition, (89%) and 22/24 (92%) horses on admission, respectively. After horses with a final diagnosis other than AM were also removal of one outlier with hypoglycaemia, blood glucose excluded. Signalment, year of hospitalisation, admission and lactate concentrations were significantly positively parameters and selected clinical signs present prior to or at correlated (q = 0.39; P = 0.011) while blood glucose and admission (increased recumbency, distension of urinary triglyceride concentrations were not correlated (q = 0.056; bladder, signs of colic), biochemical results, treatment (yes/ P = 0.41). no: intravenous glucose, intravenous insulin, vitamins Twenty-eight horses survived (44%), 30 were subjected to [including oral or intravenous administration of vitamin B, C, E euthanasia and six died (nonsurvivors 56%). Comparison of and/or multivitamins], oral carnitine and intravenous or clinicopathological data between survivors and nonsurvivors intramuscular sedation with alpha 2 agonists), days of is shown in Tables 1 and 2. Increasing CK activities and hospitalisation and outcome (from survival to discharge, triglyceride concentrations, but not AST activities during death or until being subjected to euthanasia) were recorded. hospitalisation were associated with outcome and none Horses that were subjected to euthanasia due to financial of the treatments with the exception of sedation were considerations were excluded from outcome analysis. Serum associated with outcome (Table 2). Post-mortem examination CK and AST activity and triglyceride concentrations obtained was carried out on 16/36 nonsurvivors in this study and 1/22 on subsequent days of hospitalisation were recorded and survivors had muscle biopsies. Findings were consistent with categorised as either increased or decreased. An increase or AM including multifocal process compatible with Zenker’s decrease was based on comparison of the highest value degeneration and necrosis in fibres of postural and/or measured during hospitalisation to the admission sample. respiratory muscles (Cassart et al. 2007; van Galen et al. Where available, post-mortem findings were recorded. 2012a).

Statistical analysis Discussion Data were analysed using a commercially available software programme. Normality was assessed using the Kolmogorov– The 56% mortality rate of horses suffering from AM in this study Smirnov test. Normally distributed data were recorded as was numerically lower than that documented in previous mean Æ standard deviation; non-normally distributed data studies in Europe (74–97%) (Brandt et al. 1997; Westermann were displayed as median and range. Categorical data (sex, et al. 2008; van Galen et al. 2012a) and comparable to or presence/absence of recumbency, urinary bladder distension slightly better than a recent study in the UK (61%) and colic signs, increase/decrease of CK and AST activity (Gonzalez-Medina et al. 2017). However, direct comparison and triglyceride concentrations on subsequent days) were with other studies is difﬁcult. In contrast to this study, analysed using chi-squared or Fisher’s exact test. Relevant hospitalisation was not a requirement for inclusion in previous continuous data (age at presentation, heart and respiratory studies. It is possible that more intensive support provided to rates, rectal temperature, CK and AST activities, packed cell hospitalised horses contributed to a better outcome (van volume, total protein, creatinine, glucose, lactate and Galen et al. 2012a). On the other hand, mildly or very

TABLE 1: Clinicopathological parameters (continuous data) obtained on admission to referral hospitals from horses with atypical myopathy, divided into horses surviving and not surviving to discharge. Data are presented as mean Æ standard deviation (normally distributed data) or median (range) for non-normally distributed data. A P value ≤0.05 was considered statistically signiﬁcant

Outcome

Admission parameter n Survivors Nonsurvivors Reference values P value

Age (years) 63 6 (0.25–22) 4 (0.25–18) 0.32 Heart rate (bpm) 63 60 (40–90) 61 (40–110) 0.86 Respiratory rate (bpm) 62 20 (12–44) 20 (12–66) 0.22 Temperature (°C) 54 37.5 Æ 0.6 36.9 Æ 1.00 0.006 Packed cell volume (%) 53 40 (34–57) 48 (33–73) 32–53 0.007 Total protein (g/L) 58 70 Æ 10 73 Æ 10 52–79 0.3 Calcium (mmol/L) 29 2.68 (1.37–2.93) 1.5 (0.25–3.1) 2.5–3.6 0.004 Creatinine (lmol/L) 51 98 (62–207) 117 (60–294) 106–168 0.01 Serum CK activity (U/L) 47 38,369 (7024–570,498) 172,687 (2036–570,953) 108–430 0.017 Serum AST activity (U/L) 47 4721 (676–25,368) 9666 (1070–61,782) 226–366 0.15 Triglycerides (mmol/L) 27 3.8 (0.6–21.1) 3.5 (0.1–6.6) 0.17–0.46 0.2 Blood glucose (mmol/L) 40 8.4 Æ 2.7 10.2 Æ 3.4 4.2–6.4 0.18 Lactate (mmol/L) 39 3.5 (0.5–10.4) 7.3 (2.5–16.5) <2.0 0.006 Days of hospitalisation (day) 64 7 (5–15) 2 (1–12) <0.001 severely affected horses and horses found dead might not time worsened the prognosis. As overlap between groups have been referred, and the effect on overall prognosis is was large and case numbers relatively small, CK values difficult to predict. The study provides important information should always be interpreted in light of the clinical findings nonetheless. Veterinarians might be able to advise horse and ideally also in conjunction with follow-up enzyme owners interested in referral better about expected outcome measurements. In this study, we did not observe fluctuations whilst in the past the poor prognosis might have deterred in CK activities once values had started to decrease and veterinarians and horse owners alike from referral. once a decrease was detected it tended to continue. Serum In this study, presence or absence of increased AST activity, which increases more slowly than CK activity, recumbency, colic signs or bladder distension were not was not significantly higher in nonsurvivors than survivors. It is associated with survival which is in contrast to previous possible that in nonsurviving horses, due to rapid observations (van Galen et al. 2012b; Boemer et al. 2017; deterioration, AST activity never actually increased to its full Gonzalez-Medina et al. 2017). This might be associated with extent. The significantly shorter duration of hospitalisation the retrospective nature of this study and inconsistencies in observed in nonsurvivors supports this assumption. In the the record keeping. Presence of a clinical sign tends to be future, measurements of acylcarnitine profiles might add more frequently recorded than the absence which might information that can be used prognostically (Boemer et al. explain the lack of a significant difference in regards to colic 2017). However, as it is unlikely that any single predictive signs and bladder distension. However, the latter was close to indicator or combination of predictive factors will be 100% significance. Regardless of the underlying cause, the inability accurate, decisions about continuation of treatment are in to stand is usually associated with a poor prognosis in adult the authors’ opinion best based on case progression in horses, which has also been demonstrated for AM (van combination with individual circumstances. Galen et al. 2012b; Boemer et al. 2017; Gonzalez-Medina Due to the profound effects of hypoglycin A and its toxic et al. 2017). Although increased time spent recumbent was metabolites on energy metabolism, affected horses often frequently recorded in the cases presented here, it is a present with hyperglycaemia, hyperlactataemia and increased subjective statement. It was also difficult to ascertain from plasma triglyceride concentrations. Hyperglycaemia was records whether cases were unable to rise or simply spent present in 76% of cases on admission, similar to what has been more time laying down which might explain the lack of reported before (Finno et al. 2006; Votion et al. 2007), while only significance. one horse presented with hypoglycaemia. Significant In cases of myopathy, CK release into the plasma is differences between survivors and nonsurvivors were not proportionate to the amount of muscle damaged and identified which could be associated with low case numbers or intuitively, one would expect extensive damage to be the overall common occurrence of this abnormality. However, associated with a worse outcome (Toutain et al. 1995). plasma lactate concentrations were significantly higher in Undoubtedly, the specific muscle group involved also plays a nonsurvivors compared to survivors. Previously, increased significant role as damage to myocardium or respiratory plasma lactate concentrations in sick horses have widely been muscles likely has a large impact on prognosis, even if a regarded as an indicator of anaerobic metabolism secondary relatively small amount of muscle mass is affected. The two to tissue hypoxia. The increased PCV and creatinine most recent studies have both identified an association concentrations observed here suggest that some degree of between initial CK activities and survival (Boemer et al. 2017; hypovolaemia was present in most horses, although renal Gonzalez-Medina et al. 2017) while authors of a previous damage could also have contributed to increases in the latter study observed that CK enzyme activity not always parameter (Cassart et al. 2007; van Galen et al. 2012a). More correlated closely with the deterioration of clinical signs recently, the close interactions between glucose and lactate (Votion et al. 2007). Results in the present study also indicated metabolism have been emphasised as an alternative cause of that high admission values and increasing CK activities over hyperlactataemia (Garcia-Alvarez et al. 2014a,b). Any increase

TABLE 2: Clinicopathological parameters (categorical data) from reports can probably, at least partially, be attributed to horses with atypical myopathy presenting to referral hospitals, increased glycolysis and decreased oxidative pathways divided into horses surviving and not surviving to discharge. A P- (Garcia-Alvarez et al. 2014b; Kaukonen et al. 2014). This ≤ fi value 0.05 was considered statistically signi cant assumption is supported by the positive correlation between Outcome blood glucose and lactate concentrations. Most horses in this study presented with increased plasma triglyceride Variable Survivors Nonsurvivors Total P value concentrations. As nearly all horses were admitted shortly after onset of clinical signs without any prolonged periods of Admission year starvation, this finding reflects the impact of the toxic 2011 3 4 7 (11%) 0.59 2012 1 2 3 (5%) metabolites on lipid and energy metabolism (Votion et al. 2007; 2013 4 9 13 (20%) Lemieux et al. 2016). It also highlights the importance of 2014 13 12 25 (39%) determining triglyceride concentrations in affected horses. 2015 1 5 6 (9%) While admission triglyceride concentrations were not associated 2016 1 1 2 (3%) with outcome, a further increase on subsequent measurements 2017 5 3 8 (13%) during hospitalisation was associated. In some horses, Total 28 (44%) 36 (56%) 64 measurements fluctuated, often reflecting the level of nutritional Increased recumbency prior to/at admission support (almost exclusively intravenous glucose infusions) Yes 8 (35%) 13 (43%) 21 (40%) 0.37 provided to the patient. The fact that nonsurvivors were more No 15 (65%) 17 (57%) 32 (60%) likely to have a further increase in triglyceride concentrations Total 23 30 53 Colic prior to/at admission after admission could indicate more severe metabolic Yes 4 (17%) 4 (12%) 8 (14%) 0.44 disturbances. Further studies focusing on this aspect are No 20 (83%) 30 (88%) 50 (86%) needed. Total 24 34 58 Admission temperature was lower in nonsurvivors Bladder distension prior to/at admission compared to survivors which is a fact that correlates with Yes 2 (50%) 10 (71%) 12 (67%) 0.057 findings of previous studies. This might represent reduced No 2 (50%) 4 (29%) 6 (33%) peripheral perfusion due to cardiovascular compromise or Total 4 14 18 reduced rectal tone (Votion et al. 2007; Westermann et al. Intravenous glucose 2011; van Galen et al. 2012a,b). Low serum calcium Yes 15 (54%) 17 (49%) 27 (43%) 0.69 No 13 (46%) 18 (51%) 24 (57%) concentrations have previously been reported in horses Total 28 35 63 succumbing to AM (Votion et al. 2007; Boemer et al. 2017). Intravenous insulin Calcium deposition in damaged tissues is the main cause of Yes 6 (21%) 10 (29%) 16 (25%) 0.57 hypocalcaemia in people with rhabdomyolysis and this could No 22 (79%) 25 (71%) 47 (75%) also be true in horses (Akmal et al. 1986). Other electrolyte Total 28 35 63 abnormalities less frequently recorded in the literature were Intravenous and oral vitamins (including B, C, E and multivitamins) not investigated in this study (Votion et al. 2007; van Galen Yes 24 (86%) 25 (71%) 49 (78%) 0.15 et al. 2013). Use of sedation was significantly associated with No 4 (14%) 10 (29%) 14 (22%) outcome with 62.1% of nonsurvivors receiving sedation Total 28 35 63 compared to 18.2% survivors. Sedation was used to relieve Oral carnitine Yes 12 (47%) 10 (29%) 22 (35%) 0.24 oesophageal obstruction, a common sequela of AM (Finno No 16 (53%) 25 (71%) 41 (65%) et al. 2006; Boemer et al. 2017) and in animals in pain or Total 28 35 63 distress. Both repeated oesophageal obstruction and the Intravenous or intramuscular sedation with alpha 2 agonists degree of pain are likely indicative of more extensive disease Yes 4 (17%) 20 (61%) 24 (42%) 0.001 and were associated with a worse prognosis in a recent study No 20 (83%) 13 (39%) 33 (58%) (Boemer et al. 2017). Severe pain and distress might also be Total 24 33 57 reasons for euthanasia due to welfare considerations, Increasing CK activities during hospitalisation particularly if the prognosis is perceived to be poor. Due to Yes 8 (38%) 9 (90%) 17 (55%) 0.008 their inhibitory effects on insulin release, some authors No 13 (62%) 1 (10%) 14 (45%) Total 21 10 31 consider use of alpha 2 agonists contraindicated in horses Increasing AST activities during hospitalisation with AM and a direct negative impact on survival in this study Yes 17 (81%) 10 (100%) 27 (87%) 0.19 cannot be excluded (Fabius and Westermann 2018). In other No 4 (19%) 0 4 (13%) studies, use of vitamins and/or antioxidants has been Total 21 10 31 associated with a better outcome (van Galen et al. 2012b). Increasing triglyceride concentrations activity during This was not apparent in this study, probably due to the hospitalisation overall quite uniform treatment approach (almost 80% of Yes 3 (38%) 4 (100%) 7 (58%) 0.038 horses received some form of vitamins) and small numbers. In No 5 (62%) 0 5 (42%) addition, carnitine was administered orally in all cases but Total 8 4 12 due to its low bioavailability absorption is very limited (Foster et al. 1988; Zeyner and Harmeyer 1999). The effects of in pyruvate availability, either by increased glycolysis or intravenous administration should therefore be re-evaluated decreased utilisation secondary to decreased Krebs cycle before conclusions about its efficacy are drawn. activity or decreased oxidative metabolism, can lead to The largest limitations of the present study are the increased plasma lactate concentrations. Hyperglycaemia and retrospective nature, relatively low case numbers and, hyperlactataemia observed in horses with AM in this and other particularly in regards to effects of treatment, a likely strong

selection bias. Information about certain clinical signs found Boemer, F., Deberg, M., Schoos, R., Baise, E., Amory, H., Gault, G., to be useful in other studies such as sweating, muscle Carlier, J., Gaillard, Y., Marcillaud-Pitel, C. and Votion, D. (2015) Quantification of hypoglycin A in serum using aTRAQ((R)) assay. J. fasciculations, anorexia and oesophageal obstruction, were Chromatogr. B Analyt. Technol. Biomed. Life Sci. 997, 75-80. not reliably recorded in all records and were therefore not Boemer, F., Detilleux, J., Cello, C., Amory, H., Marcillaud-Pitel, C., analysed (van Galen et al. 2012b; Boemer et al. 2017). Richard, E., van Galen, G., van Loon, G., Lefere, L. and Votion, Although a standardised data collection form was applied, D.M. (2017) Acylcarnitines profile best predicts survival in horses the details that could be obtained for each case varied with atypical myopathy. PLoS ONE 12, e0182761. and little follow-up information was available. However, AM Brandt, K., Hinrichs, U., Glitz, F., Landes, E., Schulze, C., Deegen, E., remains a sporadic disease and obtaining large case Pohlenz, J. and Coenen, M. (1997) Atypische Myoglobinurie der numbers from a certain region or a single country remains Weidepferde. Pferdeheilkunde 13, 27-34. difficult. Although advances in identification of toxic Cassart, D., Baise, E., Cherel, Y., Delguste, C., Antoine, N., Votion, D., metabolites responsible for AM have been made, those Amory, H., Rollin, F., Linden, A., Coignoul, F. and Desmecht, D. (2007) Morphological alterations in oxidative muscles and methods have only recently become more widely available mitochondrial structure associated with equine atypical myopathy. (Boemer et al. 2015; Sander et al. 2016). In the majority of Equine Vet. J. 39, 26-32. fi clinical cases, diagnosis of AM still relies on identi cation of Fabius, L.S. and Westermann, C.M. (2018) Evidence-based therapy for a severe myopathy in horses at pasture with sycamore atypical myopathy in horses. Equine Vet. Educ. 30, 616-622. trees being found in the vicinity. It is therefore possible that Finno, C.J., Valberg, S.J., Wunschmann, A. and Murphy, M.J. (2006) some horses in this report could have suffered from a Seasonal pasture myopathy in horses in the midwestern United different myopathy. Considering that AM is by far the most States: 14 cases (1998–2005). J. Am. Vet. Med. Assoc. 229, 1134- common cause of myopathies at pasture, most, if not all, 1141. horses were probably correctly diagnosed. This was Foster, C.V., Harris, R.C. and Snow, D.H. (1988) The effect of oral L-carnitine supplementation on the muscle and plasma supported by post-mortem findings which were strongly concentrations in the Thoroughbred horse. Comp. Biochem. suggestive of AM. The authors believe that the study, Physiol. A Comp. Physiol. 91, 827-835. despite these limitations, still provides useful information for van Galen, G., Marcillaud Pitel, C., Saegerman, C., Patarin, F., Amory, practitioners, particularly in the UK, when dealing with these H., Baily, J.D., Cassart, D., Gerber, V., Hahn, C., Harris, P., Keen, difficult cases. J.A., Kirschvink, N., Lefere, L., McGorum, B., Muller, J.M., Picavet, In conclusion, this study suggests that the mortality rate in M.T., Piercy, R.J., Roscher, K., Serteyn, D., Unger, L., Van Der Kolk, J.H., van Loon, G., Verwilghen, D., Westermann, C.M. and Votion, hospitalised horses suffering from AM could be better than D.M. (2012a) European outbreaks of atypical myopathy in grazing previously reported, and that admission serum CK activity equids (2006–2009): spatiotemporal distribution, history and clinical could be of prognostic value. A larger study determining features. Equine Vet. J. 44, 614-620. mortality rates of hospitalised and nonhospitalised patients is van Galen, G., Saegerman, C., Marcillaud Pitel, C., Patarin, F., Amory, required in order to assess whether these results represent true H., Baily, J.D., Cassart, D., Gerber, V., Hahn, C., Harris, P., Keen, improvement in mortality rate. In addition, metabolic J.A., Kirschvink, N., Lefere, L., McGorum, B., Muller, J.M., Picavet, M.T., Piercy, R.J., Roscher, K., Serteyn, D., Unger, L., van Der Kolk, abnormalities are frequent in horses with AM and require J.H., Van Loon, G., Verwilghen, D., Westermann, C.M. and Votion, further investigations. D.M. (2012b) European outbreaks of atypical myopathy in grazing horses (2006-2009): determination of indicators for risk and Authors’ declaration of interests prognostic factors. Equine Vet. J. 44, 621-625. van Galen, G., Cerri, S., Porter, S., Saegerman, C., Lefere, L., Roscher, No conflicts of interest have been declared. K., Marr, C., Amory, H. and Votion, D.M. (2013) Traditional and quantitative assessment of acid-base and shock variables in horses with atypical myopathy. J. Vet. Intern. Med. 27, 186-193. Ethical animal research Garcia-Alvarez, M., Marik, P. and Bellomo, R. (2014a) Sepsis-associated Ethical approval was granted by the Ethics and Welfare hyperlactatemia. Crit. Care 18, 503. Committee of the Royal Veterinary College, London Garcia-Alvarez, M., Marik, P. and Bellomo, R. (2014b) Stress hyperlactataemia: present understanding and controversy. Lancet (approval number 2015/S116). Diabetes Endocrinol. 2, 339-347. Gonzalez-Medina, S., Ireland, J.L., Piercy, R.J., Newton, J.R. and Votion, Source of funding D.M. (2017) Equine atypical myopathy in the UK: epidemiological characteristics of cases reported from 2011 to 2015 and factors None. associated with survival. Equine Vet. J. 49, 746-752. Hosie, B.D., Gould, P.W., Hunter, A.R., Low, J.C., Munro, R. and Wilson, H.C. (1986) Acute myopathy in horses at grass in east and south Authorships east Scotland. Vet. Rec. 119, 444-449. B. Dunkel and A. Ryan contributed to study design, study Joskow, R., Belson, M., Vesper, H., Backer, L. and Rubin, C. (2006) execution, and data analysis and interpretation. E. Haggett Ackee fruit poisoning: an outbreak investigation in Haiti 2000–2001, and review of the literature. Clin. Toxicol. (Phila) 44, 267- and E. J. Knowles contributed to data analysis and 273. interpretation. All authors contributed to the preparation of fi Kaukonen, K.M., Bailey, M., Egi, M., Orford, N., Glassford, N.J., the manuscript and gave their nal approval. Marik, P.E. and Bellomo, R. (2014) Stress hyperlactatemia modifies the relationship between stress hyperglycemia and outcome: a retrospective observational study. Crit. Care Med. References 42, 1379-1385. Akmal, M., Bishop, J.E., Telfer, N., Norman, A.W. and Massry, S.G. Lemieux, H., Boemer, F., van Galen, G., Serteyn, D., Amory, H., Baise, (1986) Hypocalcemia and hypercalcemia in patients with E., Cassart, D., van Loon, G., Marcillaud-Pitel, C. and Votion, D.M. rhabdomyolysis with and without acute renal failure. J. Clin. (2016) Mitochondrial function is altered in horse atypical Endocrinol. Metab. 63, 137-142. myopathy. Mitochondrion 30, 35-41.

Meda, H.A., Diallo, B., Buchet, J.P., Lison, D., Barennes, H., Ouangre, (2009) Atypical myopathy in grazing horses: a first exploratory data A., Sanou, M., Cousens, S., Tall, F. and Van De Perre, P. (1999) analysis. Vet. J. 180, 77-87. Epidemic of fatal encephalopathy in preschool children in Burkina Votion, D.M., van Galen, G., Sweetman, L., Boemer, F., De Tullio, P., Faso and consumption of unripe ackee (Blighia sapida) fruit. Dopagne, C., Lefere, L., Mouithys-Mickalad, A., Patarin, F., Lancet 353, 536-540. Rouxhet, S., Van Loon, G., Serteyn, D., Sponseller, B.T. and Valberg, Palencia, P. and Rivero, J.L. (2007) Atypical myopathy in two grazing S.J. (2014) Identification of methylenecyclopropyl acetic acid in horses in northern Spain. Vet. Rec. 161, 346-348. serum of European horses with atypical myopathy. Equine Vet. J. 46, 146-149. Sander, J., Cavalleri, J.M., Terhardt, M., Bochnia, M., Zeyner, A., Zuraw, A., Sander, S., Peter, M. and Janzen, N. (2016) Rapid diagnosis of Westermann, C.M., Dorland, L., Votion, D.M., De Sain-Van Der Velden, hypoglycin A intoxication in atypical myopathy of horses. J. Vet. M.G., Wijnberg, I. D., Wanders, R.J., Spliet, W.G., Testerink, N., Diagn. Invest. 28, 98-104. Berger, R., Ruiter, J.P. and Van Der Kolk, J.H. (2008) Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with Sponseller, B.T., Valberg, S.J., Schultz, N.E., Bedford, H., Wong, D.M., Kersh, K. and Shelton, G.D. (2012) Equine multiple acyl-CoA atypical myopathy. Neuromuscul. Disord. 18, 355-364. dehydrogenase deficiency (MADD) associated with seasonal Westermann, C.M., Dorland, L., Van Diggelen, O.P., Schoonderwoerd, pasture myopathy in the midwestern United States. J. Vet. Intern. K., Bierau, J., Waterham, H.R. and Van Der Kolk, J.H. (2011) Med. 26, 1012-1018. Decreased oxidative phosphorylation and PGAM deficiency in Toutain, P.L., Lassourd, V., Costes, G., Alvinerie, M., Bret, L., horses suffering from atypical myopathy associated with acquired Lefebvre, H.P. and Braun, J.P. (1995) A non-invasive and MADD. Mol. Genet. Metab. 104, 273-278. quantitative method for the study of tissue injury caused by Westermann, C.M., Van Leeuwen, R., Van Raamsdonk, L.W. and Mol, intramuscular injection of drugs in horses. J. Vet. Pharmacol. H.G. (2016) Hypoglycin A concentrations in maple tree species in Ther. 18, 226-235. the Netherlands and the occurrence of atypical myopathy in Votion, D.M., Linden, A., Saegerman, C., Engels, P., Erpicum, M., Thiry, horses. J. Vet. Intern. Med. 30, 880-884. E., Delguste, C., Rouxhet, S., Demoulin, V., Navet, R., Sluse, F., Whitwell, K.E., Harris, P. and Farrington, P.G. (1988) Atypical Serteyn, D., van Galen, G. and Amory, H. (2007) History and myoglobinuria: an acute myopathy in grazing horses. Equine Vet. clinical features of atypical myopathy in horses in Belgium (2000– J. 20, 357-363. 2005). J. Vet. Intern. Med. 21, 1380-1391. Zeyner, A. and Harmeyer, J. (1999) Metabolic functions of L-carnitine Votion, D.M., Linden, A., Delguste, C., Amory, H., Thiry, E., Engels, P., and its effects as feed additive in horses. A review. Arch. van Galen, G., Navet, R., Sluse, F., Serteyn, D. and Saegerman, C. Tierernahr. 52, 115-138.

Original Article Evaluation of a pinhole castration technique in ponies: Comparing single with double ligation (using silk or catgut) of the spermatic cord M. Y. Mir†, M. R. Fazili‡* , K. H. Dar†, M. S. Mir§ and S. Qureshi¶ †Division of Veterinary Surgery and Radiology, Faculty of Veterinary Sciences and AH, Shere Kashmir University of Agricultural Sciences and Technology of Kashmir; ‡Veterinary Clinical Services Complex, Faculty of Veterinary Sciences and AH, Shere Kashmir University of Agricultural Sciences and Technology of Kashmir; §Division of Veterinary Pathology, Faculty of Veterinary Sciences and AH, Shere Kashmir University of Agricultural Sciences and Technology of Kashmir; and ¶Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and AH, Shere Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, Kashmir, India *Corresponding author email: [email protected]

Keywords: horse; castration; catgut; pinhole; ponies; silk; spermatic cord

Summary Schumacher 1992, 1996). The orchiectomy procedure is The objective of the study was to evaluate a pinhole expensive for the poor owners (of working ponies) residing in castration technique in male ponies. Adult ponies (n = 12) the developing nations. Being invasive, strict asepsis and were randomly allotted to one of the two equal groups. Both trained surgeons are required (Hafez 1993; Trotter 1988). Post- of the groups were anaesthetised with xylazine–ketamine– operative care during second intention healing of the scrotal diazepam and had single (SLS, SLC) ligation of the spermatic wounds is also essential (Searle et al. 1999). The pain and stress cord on one side and double (DLS, DLC) ligation on the other associated with this surgery may reduce the immunity of the side using silk (Group T1) and catgut (Group T2). Single animals. Laparoscopic orchiectomy is a minimally invasive ligation whether using silk or catgut (SLS and SLC) was procedure but requires expensive instruments and special completed in 3.50 Æ 0.34 min. DLS took 6.66 Æ 0.49 min and expertise (Hendrickson 2012). Inadvertent perforation of DLC 7.16 Æ 0.47 min. Scrotal oedema was noticed in all of the viscera or blood vessels is the major disadvantage (Ragle ponies from Day 1 to Day 15. The scrotal circumference and et al. 1998; Desmaizieres et al. 2003). Immunologic castration testicular volume in animals of both of the groups showed a using a vaccine against Gonadotropin Releasing Hormone significant (P<0.05) increase from Day 1 to Day 4. Orchiectomy (GnRH) that suppresses testicular function of the stallion has to recover testicular remnants was performed in all of the been described (Malmgren et al. 2001). However, these horses animals on Day 41. Straw-coloured fluid accumulation was become sterile temporarily and repeated immunisation is noticed in seven testicular remnants (4 T1 and 3 T2). One needed. The response to vaccination also varies from animal testicle from Group DLS showed suppuration. Multiple gross to animal and their libido does not get abolished (Janett et al. and histological abnormalities were detected in all double 2009). Section-Ligation-Release (SLR) technique wherein the ligated testes. The changes were more severe in the DLS than spermatic cords are triple clamped, transected and ligated, the DLC group and their epididymis (n = 3) also showed without removing the testes has been successful in castrating necrosis, fibroplasia and an obstructed lumen. Severe limited numbers of horses (Saifzadeh et al. 2008). Being adhesions had developed in three and one testicle only from invasive in nature the procedure may also be accompanied the SLS and SLC groups respectively. The remaining testes and by several limitations of the conventional orchiectomy. epididymis in both of these groups showed only mild-to- Development of an alternative simple, inexpensive and moderate adhesions. From this study, it was concluded that reliable procedure of equine castration is therefore needed castration may not be achieved by percutaneous single particularly for application under field conditions in the ligation of the spermatic cord in ponies. Although double developing countries. Pinhole castration (in situ spermatic ligation induces marked gross and histopathological changes, cord ligation) has been described as a novel minimally assessments of the testosterone levels and sperm analysis are invasive technique in ruminants (Ponvijay 2007; Okwee-Acai required before recommending this procedure. Use of silk for et al. 2008; Fazili et al. 2009) and stray dogs (Baba et al. ligation of the spermatic cord is advantageous over catgut 2013). In donkeys, the technique was satisfactorily used but maintenance of strict asepsis is mandatory. (Badawy 2009) in one study. However, in another trial it failed to induce necrosis and atrophy of the testes (Abu-Ahmed et al. 2012). Considering the highly optimistic reports in farm Introduction animals and dogs but contradictory initial results in equids, a Bilateral orchiectomy (orchidectomy) is the most popular prospective study was planned to evaluate a pinhole technique to castrate male horses (Schumacher 2012). The castration technique using silk or catgut for single or double procedure ensures sterilisation but is associated with several ligation of the spermatic cords in male ponies. disadvantages and complications in 20–38% of horses (Mason et al. 2005). They include haemorrhage, eventration, excessive Materials and methods post-operative swelling, septic funiculitis, champignon, hydrocele, toxaemia, malignant oedema, tetanus and Twelve healthy, adult, male ponies used as pack animals were peritonitis (Van-Der-Velden and Rutgers 1990; Hunt 1991; included in the study after a written consent was obtained

individually from their owners. The project was also approved by the institutional post-graduate student advisory committee. Every animal was subjected to a detailed clinical examination including conﬁrmation of normal bilateral descent of the testes. They were dewormed with albendazole (7.5 mg/kg bwt per os) and injected tetanus toxoid (0.5 mL/50 kg, i.m.) 7 days prior to the initiation of the study. Ponies were randomly allotted to one of the two equal groups (Fig 1). The animals were designated for percutaneous ligation of the spermatic cord (pinhole castration) using silk (Group T1) or catgut (Group T2), respectively, as per the technique described earlier (Ponvijay 2007; Fazili et al. 2009). Xylazine–ketamine–diazepam combination as total i.v. anaesthesia (TIVA) was used to induce general anaesthesia in animals that had been fasted overnight. In the laterally recumbent animals, and after pushing the hindlimbs cranially, the scrotal area was scrubbed using 4% chlorhexidine scrub solution diluted with 0.9% sterile normal Fig 2: Percutaneous spermatic cord ligation (Pinhole castration) saline. In ponies of Group T1, a reverse cutting (traumatic) underway in a pony using a traumatic needle threaded with silk. stainless steel suture needle threaded with braided silk (No. 2) was used for the formation of a suture loop around the spermatic cord (Fig 2). A square knot was applied and the extra thread trimmed 3.0 mm proximal to the knot. For double ligation of the spermatic cord, the procedure was repeated 1.5–2.0 cm distal to the ﬁrst ligation. Povidone Iodine (0.5%) was subsequently applied to the puncture site. In animals of Group T2, the technique of spermatic cord ligation was similar to that of Group T1 except that chromic catgut (No. 2) was passed through a G-16 hypodermic needle for ligation (Fig 3). In all of the ponies, one testis was subjected to single ligation (SLS or SLC) and the contralateral to double ligation (DLS or DLC) of the spermatic cord. Thus, a total of six testes in each group (T1 and T2) were subjected to single ligation and the remaining six to double ligation. Time required for the procedure was recorded. Penicillin– streptomycin (2.5 g, i.m.) and meloxicam (0.4 mg/kg bwt, i.v.) were given to all of the animals for 3 days. The ponies were Fig 3: Percutaneous spermatic cord ligation (Pinhole castration) maintained by their owners as per routine and were not put underway in a pony using a hypodermic needle to pass chromic to work throughout the study period due to the prevailing off- catgut. season. They were clinically examined daily up to Day 4 and again on Days 8, 15, 28 and 40. Circumference of scrotum, in situ length (L), width (W) under xylazine–ketamine anaesthesia on Day 41 post-ligation and height (H) of the testis were recorded on Days 0, 1, 4, 8, and weighed. The recovered testicles and epididymis were 15, 28 and 40. The testicular volume was calculated using a thoroughly examined for gross alterations and then preserved standard formula L 9 W 9 H 9 0.5236 (Love et al. 1991). in 10% formalin for histopathological evaluation. The testes/remnants from all the animals were recovered The ponies were turned out in small paddocks and by a conventional orchiectomy (open method) procedure monitored daily. All of them recovered without complications.

PONIES 12 (spermatic cords 24)

GROUPS T1 SILK (n 6, cords 12) T2 CATGUT (n 6, cords 12)

LIGATION Single T1(SLS) Double T1(DLS) Single T2 (SLC) Double T2 (DLC) 6 cords 6 cords 6 cords 6 cords

Fig 1: In situ spermatic cord ligation (pinhole castration) protocol in ponies.

The statistical analysis was conducted using SPSS software- The swelling was soft (fluid) initially. After Day 15, the scrotal 15. The data were analysed using repeated measurement palpation revealed firm consistency in most (n = 8) of the ANOVA (Grimm and Yarnold 2000). Students T test was used animals of both groups. In one pony of Group T1, the wherever two groups were under comparison. Values with persistently swollen testicle yielded straw-coloured fluid on P<0.05 were regarded as significant. centesis by Day 25. Fistulous tract developed at the site of ligation in two of Results the ponies belonging to Group T1 on Day 10. One of these animals was successfully managed with local treatment and

Mean (Æs.e.) age of the ponies belonging to Group T1 and T2 a course of parentral antibiotic (amoxicillin–salbactum, was 6.33 Æ 0.61 years and 5.33 Æ 0.55 years, and their body 10 mg/kg bwt, i.v., b.i.d., 5 days). The second pony did not weight 211.50 Æ 13.20 kg and 209.33 Æ 8.94 kg respectively. respond to the treatment until orchiectomy. No significant (P>0.05) difference was found in their age and The scrotal circumference and in situ volume (mean Æ s.e.) body weight. of the testicles in ponies belonging to Groups T1 and T2 are The castrations were performed outdoors on level grassy shown in Tables 1 and 2 respectively. ground. However, due to the inclement weather, three The circumference of the scrotum increased from Day 1 to animals were castrated inside a shed over straw bedding. Day 4 and showed a decreasing trend thereafter in ponies of Anaesthesia was satisfactory, lasted for an adequate both groups. The maximum (47.10 Æ 3.10 cm and 48.40 Æ period, terminated rapidly and recovery was free from 1.05 cm respectively) circumference was recorded on Day 4. excitement in all of the ponies. The minimum (41.00 Æ 2.55 cm and 31.75 Æ 0.62 cm) The spermatic cords were easily located within the circumference was recorded on Day 0 and Day 40 scrotum. Their lateral displacement posed some difficulty but respectively. In animals of Group T1, the increase in the scrotal could be held with the thumb and the fore-finger of left hand circumference showed no significant (P>0.05) difference firmly pressed together medial to the cords. Penetration of the throughout the study period. However, in those of the Group hypodermic or suture needle through and through the scrotal T2, the values obtained from Day 1 to Day 8 were significantly sac did not pose difficulty. However, on one occasion while (P<0.05) higher than the values recorded on Day 0 and again passing the hypodermic needle, straw coloured fluid dribbled Day 15 onwards. Also the value obtained on Day 4 was from its hub indicating puncture of the testicular tunic. The significantly (P<0.05) higher than that recorded on Day 8. needle was therefore withdrawn and redirected away from Between the groups the values differed significantly (P<0.05) on the testicle. The square knot got buried either automatically or Day 40 only. The value was higher in animals of Group T1 than after the spermatic cord was pushed medially (percutaneously) that of Group T2. in all Group T1 animals. However, four of the eighteen ligatures The testicular volume in animals of both groups (T1 and applied in ponies belonging to Group T2, required a gentle T2) showed significant (P<0.05) increase on Day 1 and Day 4 push using the tip of a curved mosquito artery forceps for when compared with the Day 0 value. The volume reduced complete burial. The needle holes were faintly visible at the thereafter in single ligated testes (SLS and SLC). However, in completion of the procedure (Fig 4) and most of them could the double ligated testes (DLS and DLC) swelling was again not be identified on subsequent examinations. In one of these noticed on Day 15 and Day 28 respectively. Between the animals, the ligature broke inadvertently while tying the knot. groups the testicular volume did not differ significantly The procedure was therefore repeated in this case. (P>0.05) on most of the intervals. Single ligation (SLS and SLC) of the spermatic cord in On recovery of the remnants from the opened scrota most ponies of both of the Groups was completed in of the ponies of both the groups were found to have 3.5 Æ 0.34 min. Double ligation procedure in Group DLS took developed moderate-to-severe adhesions between the 6.66 Æ 0.49 min and in Group DLC 7.16 Æ 0.47 min. testicular tunics (Fig 5) and the scrotal covering at and beyond Mild-to-moderate swelling in the scrotum was detected the ligation site. Straw-coloured fluid that had accumulated in on visualisation in all ponies of Group T1 and T2 from Day 1 to the dead space drained out while incising tunics in seven Day 15. In two of the Group T1 and three of the Group T2 ponies (4 T1 and 3 T2). One pony belonging to the DLS group animals, the prepuce also showed mild swelling up to Day 8. (that had developed a fistula) showed suppuration. The weight (mean Æ s.e.) of the testicles/remnants recovered by orchiectomy on Day 41 of the study in ponies of Group SLS and DLS was 181.66 Æ 26.09 g and 173.33 Æ 28.68 g respectively. The testes from Group SLC and DLC animals were 156.83 Æ 5.46 g and 147.16 Æ 9.13 g respectively. The values showed no significant (P>0.05) difference between the groups. However, the mean weight of the testicles of Group SLC and DLC was slightly lower than the Group T1. Furthermore, the weight of the testicles with double ligated spermatic cord in both the groups was lower than those subjected to single ligation. Gross examination revealed adhesions and haemorrhages over the tunics and capsule of all of the recovered testes/ remnants. Testes from Group DLS showed maximum gross abnormalities such as severe adhesions (5/6), diffusely spread Fig 4: Completed spermatic cord ligation showing pinhole with haemorrhagic spots (4/6), necrotic areas (4/6) and oedema submerged knot. (3/6) (Fig 6). Two testes were firm on palpation and two more

TABLE 1: Circumference of scrotum (Mean Æ s.e.) in ponies following pinhole castration

Measurements at different intervals (Days)

Parameter Group 0148152840

Circumference T1 41.00 Æ 2.55 45.50 Æ 2.92 47.10 Æ 2.57 44.46 Æ 3.41 47.75 Æ 3.15 41.76 Æ 3.15 41.41 Æ 3.35B (cm) (n = 6) T2 35.21 Æ 0.66a 45.16 Æ 0.40bc 48.40 Æ 1.00c 42.31 Æ 2.26b 34.42 Æ 1.28a 34.42 Æ 1.28a 31.75 Æ 0.62aA (n = 6)

Values with different superscripts of lower- and upper-case alphabets indicate signiﬁcant differences within and between groups respectively.

were soft with brownish discoloration. Testes recovered from The second ligation was always placed distal to the first. Group DLC ponies were also badly affected with severe This was done so that no inadvertent punctures of the adhesions (3/6), oedema (3/6), diffuse haemorrhage (2/6), firm engorged blood vessels occur. In the ponies included in this consistency (2/6) and necrotic areas (1/6). Severe adhesions study, the spermatic cord of one side was subjected to single were evident in three of the testicles from Group T1 (SLS). The ligation and the contralateral side to double ligation using remaining three had mild-to-moderate adhesions only. One the same suture material on both sides. During evaluation of testicle also showed oedema. Group T2 (SLC) testes were a new research protocol, two separate treatments are least affected and had developed adhesions (severe 2/6, preferably applied to the contralateral organs to minimise the moderate 3/6 and mild 1/6) only. bias between individuals. Double ligation of the same Adhesions were detected in the epididymis of all of the spermatic cord has to date not been performed in any testes from Group T1 and Group T2 animals. The lumen of the species. Single ligation unilaterally or bilaterally has been three epididymides recovered from Group DLS ponies also undertaken in donkeys (Badawy 2009; Abu-Ahmed et al. had obstructed lumen. 2012). On histopathological examination, complete necrosis of The holes created due to the passage of G-16 the germinal epithelium and Leydig cells along with interstitial hypodermic and the reverse-cutting suture needles were oedema (Fig 7) was detected in five of the six testicular tissues large enough to accommodate the square knot but not the recovered from ponies belonging to Group DLS. Fibroplasia surgeon knot. Use of a reverse-cutting needle reduces the risk and typical inflammatory changes were also noted in three of suture material tearing through the wound edge (Kudur and two testicles respectively. The epididymis (3/6) also et al. 2009). Chromic catgut and monofilament showed necrosis, fibroplasia and eosinophilic casts (Fig 8). polydioxanone have been used for percutaneous ligation of Interstitial oedema (5/6), complete necrosis of the germinal the spermatic cord or epididymis in animals (Badawy 2009; epithelium and Leydig cells (2/6) were also detected in the Tamadon et al. 2010; Abid and Baghdady 2013; El-Wahed testes recovered from Group DLC. The testicles in Group SLS et al. 2014). Silk being cheap and easily available was tried in showed mild-to-moderate degeneration of the germinal Group T1 ponies. Being less stiff than catgut, silk does not pass epithelium (3/6), Leydig cell necrosis (1/6) and fibroplasia (1/6). easily through a hypodermic needle making use of a suture Testicles from Group SLC were least affected and had mild-to- needle necessary (Fazili et al. 2009; Abu-Ahmed et al. 2012; moderate degeneration of the germinal epithelium (3/6). Baba et al. 2013). The mean time required to complete single ligation of the Æ Discussion spermatic cord was similar (3.5 0.34 min) in ponies whether silk or catgut was used for pinhole castration. This time period The age and the weight of the ponies included in the study was similar to the two earlier reports pertaining to donkeys varied but the mean (Æs.e.) values in both the groups were (Badawy 2009; Abu-Ahmed et al. 2012). The time required for almost similar. The ponies included in our study belonged to pinhole castration was significantly (P<0.05) less than the rural farmers who get them castrated only after attaining incision–ligation and segment–ligation–resection procedures adult age. Horses may preferably be castrated at 1–2 years (Abu-Ahmed et al. 2012). A comparatively shorter time of age beyond which objectionable masculine behaviours (40–60 s per ligation) was reported in calves (Ponvijay 2007). develop (Schumacher 2012). The spermatic cord in equines is short and thin as compared The xylazine–ketamine–diazepam combination provided with ruminants. Consequently, repeated attempts may be effective and safe anaesthesia in all of the ponies included in required to firmly grasp it. This species-specific anatomical the study. Combination of ketamine and one of the alpha-2 peculiarity may be responsible for the comparatively longer agonists is a common anaesthetic protocol followed in time required for ligation in equids. ponies under field conditions. Addition of diazepam enhances During the first week following spermatic cord ligation skeletal muscle relaxation (Mathew and Hartsfield 1993). irrespective of the suture material used and whether single or Tranquilisation/sedation (acepromazine/chloral hydrate) double ligation applied, the testicular volume increased two followed by infiltration 2% lignocaine hydrochloride locally in to three times. The volume decreased persistently thereafter. donkeys has been used satisfactorily (Badawy 2009; Abu- The values of single ligated testes obtained on Day 40 were Ahmed et al. 2012). lower than the preligated values, whereas in double ligated The spermatic cords were easily palpated within the testes, the volume at the end of the study was slightly more scrotal neck. They are slippery and slender but could be held than that of pre-operative value. This might have been due without difficulty due to the comparatively thin equine skin. to accumulation of fluid within the tunics and parenchyma

© 2018 EVJ Ltd 100 EQUINE VETERINARY EDUCATION / AE / FEBRUARY 2020 a a a a 4.96 18.05 7.73 13.83 40 Æ Æ Æ Æ 59.22 84.35 73.42 92.69 a a a a 5.76 17.90 7.59 14.09 28 Æ Æ Æ Æ 60.80 85.05 79.63 94.90 aA bB bcB aA 37.77 43.65 7.98 11.00 15 Æ Æ Æ Æ respectively. 64.42 71.50 187.93 204.95 groups (Days) Fig 5: Adhesions between the testicular tunics in a pinhole castrated pony. catgut. aA abAB bcB abAB intervals between with 13.14 45.72 39.31 43.14 and 8 Æ Æ Æ Æ different within ligation at 86.31 153.90 209.43 175.33 double bA bAB cAB cB differences DLC, Measurements 33.15 38.44 27.66 60.86 cant 4 ﬁ Æ Æ Æ Æ catgut; signi 163.85 188.18 286.40 299.48 with indicate castration b b bc bc ligation 17.62 40.23 33.65 20.22 single 1 Æ Æ Æ Æ pinhole alphabets SLC, 163.39 236.44 245.94 219.10 silk; following with upper-case a a a a ponies and 8.70 10.62 7.18 5.34 in 0 ligation Æ Æ Æ Æ s.e.) lower- Æ of 85.71 81.77 87.15 72.09 double Fig 6: Testicular remnant recovered from a pinhole castrated pony showing haemorrhagic and necrotic areas on gross DLS, (Mean examination. silk; superscripts Group T1(SLS) testes T1(DLS) T2(SLC) T2(DLC) with

of that was noticed during orchiectomy, gross and histopathological examinations. ) 3 different Swelling in the scrotum was noticed within 24 h in animals ligation

Volume ﬁ

(cm of both groups. The swelling continued to the end of the rst with

2: week and subsequently it started subsiding. The swelling also single extended up to the prepuce in some animals. The results of TABLE SLS, Values Parameter Volume an earlier study involving donkeys indicate that the prepucial

within testicular remnants was found in several ponies included in this study. Fluid accumulation has also been noticed by the authors in bucks. The severity of the histopathological changes in the testes like fibroplasia and necrosis of the germinal epithelium and Leydig cells along with interstitial oedema also corresponded well with the gross lesions. Testis with ligation of the spermatic cord showed degenerative changes indicative of ischaemic necrosis (El-Wahed et al. 2014). The abnormalities in the testicular and epididymal tissues were comparatively more severe in Group T1 than Group T2 ponies. Catgut a natural fibre absorbable suture has a tendency to fray during knot construction. It also shows considerable variation in the retention of tensile strength and rate of absorption (Williams 1998). Premature absorption of catgut is noticed in acidic and infected environments and also in highly vascularised tissues (Boothe 1985). In this study, silk was used in a total of 18 ligations in six Fig 7: Photomicrograph of testis showing marked degeneration ponies. Unilateral fistula formation in two of these ponies may of germinal epithelium in a pinhole castrated pony. be attributed to the capillarity nature of the braided silk (Kudur et al. 2009). Due to the inclement weather, the procedure in both of these animals was undertaken inside a shed. Strict asepsis is mandatory for the pinhole procedure (Ponvijay 2007). The fistula healed following antimicrobial administration in one animal but persisted in another. Inflammatory changes were also noticed in two testes on histological examination. To manage such animals orchiectomy is indicated. Necrosis of the germinal epithelium and Leydig cells along with interstitial fibrosis noticed in the testicular tissue are valid indications of a successful castration (Abu-Ahmed et al. 2012). Interstitial oedema and suppurative inflammation are two undesirable findings (Abu-Ahmed et al. 2012). Epididymal epithelium of Group DLS animals had undergone degenerative changes with accumulation of necrotic material within their lumens. This finding was also observed in dogs (Baba et al. 2013). The complete absence of spermatozoa in the epididymis in Group (DLS) is an important sign of castration. Fig 8: Photomicrograph of epididymis showing eosinophilic Interestingly the testicles with single ligation of the material within lumen, degeneration of epithelium and fibroplasia spermatic cord in ponies of both groups included in this study in a pinhole castrated pony. showed comparatively mild gross and histopathological alterations, indicating uncertain castration. Laparoscopic circumference and length increased significantly up to Day 3 castration performed in stallions had a failure rate 3.4–12.5%, in the incision–ligation but not in pinhole group (Abu-Ahmed wherein complex blood supply has been suggested as its et al. 2012). Moderate swelling of the ligated testicles for 3– cause (Voermans et al. 2006; de-Fourmestraux et al. 2014). 4 days in donkeys (Badawy 2009; Abu-Ahmed et al. 2012) Blood vessels like cremasteric or external pudental artery or and up to 6 days in calves (Ponvijay 2007) has been noticed. both can revascularise the testis. This peculiarity of the equine In donkeys the scrotal swelling began to decrease from Day testicular blood supply may substantiate the reported success 10 to Day 12 and reached to the baseline measurements of incision–ligation and section–ligation–release techniques about 18 days post-ligation (Badawy 2009; Abu-Ahmed et al. (involving a segment of the spermatic cord thus reducing the 2012). chances of revascularisation) but failure of the pinhole The scrotal circumference was pre-operatively (Day 0) procedure (with single ligation of both the spermatic cords) in nonsignificantly less in ponies of Group T2 than those of sterilisation of donkeys (Abu-Ahmed et al. 2012). Better results Group T1. The difference was significant (P<0.05) on the last in ponies with double ligation of the spermatic cords (than recording (Day 40) due to the suppurative inflammation in those with single ligation) obtained in this trial may also be two testicles of Group T1 ponies. attributed to the same peculiarity. While performing orchiectomy on Day 41, adhesions The weaknesses of this trial include estimation of blood between the tunics and the scrotal skin were detected in all testosterone level, sperm production status and the long-term of the animals. Adhesion formation following pinhole effect on the sexual behaviour of the equines subjected to castration has also been previously reported in many species pinhole castration. The costs associated with the pinhole (Ponvijay 2007; Badawy 2009; Abid and Baghdady 2013; procedure and its comparison to that of traditional Baba et al. 2013). The straw-coloured fluid accumulation orchiectomy have also not been calculated.

Conclusions techniques used for laparoscopy in standing horses. Vet. Surg. 32, 501-506. From this study, it was concluded that castration may not be El-Wahed, R.E.A., Korittum, A.S., Abu-Ahmed, H.M. and Sahwan, A.A.S. achieved by percutaneous single ligation of the spermatic (2014) Evaluation of pinhole castration technique compared with cord in ponies. Although double ligation induces marked traditional method for castration in dogs. Alex. J. Vet. Sci. 42, 90-98. gross and histopathological changes, assessment of the Fazili, M.R., Bhattacharyya, H.K., Buchoo, B.A., Kirmani, M.A., Darzi, testosterone level following double bilateral ligation of the M.M. and Khan, I. (2009) Evaluation of pinhole castration spermatic cord is required before recommending this technique in rams. Small Rumin. Res. 84, 61-64. procedure. Use of silk for ligation of the spermatic cord is de-Fourmestraux, C., Geffroy, O., Siliart, B., Albaric, O and Tessier, C. preferable to catgut but maintenance of strict asepsis is (2014) Evaluation of success rate of laparoscopic castration without orchidectomy in 32 mature horses. Equine Vet. Educ. 26, mandatory. 34-39. Grimm, L.G. and Yarnold, P.R. (2000) Reading and Understanding Authors’ declaration of interests Multivariate Statistics, American Psychological Association, Washington, DC. pp 380-391. No conflicts of interest have been declared. Hafez, E.S.E. (1993) Reproduction in Farm Animals, 6th edn., Lea & Febiger, Philadelphia. pp 140-144. Hendrickson, D.A. (2012) A review of equine laparoscopy. ISRN Ethical animal research Veterinary Science Article ID 492650, 17 pages, https://doi.org/10. Written consent for this minimally invasive procedure was 5402/2012/492650 obtained from all the pony owners individually before Hunt, R.J. (1991) Management of complications associated with equine inclusion of their animals in the study. The project was also castration. Compend. Contin. Educ. Pract. Vet. 13, 1835-1873. approved by the institutional post-graduate student advisory Janett, F., Stump, R., Burger, D. and Thun, R. (2009) Suppression of committee. testicular function and sexual behaviour by vaccination against GnRH (Equity™) in the adult stallion. Anim. Reprod. Sci 115, 88-102. Kudur, M.H., Pal, S.B., Sripathi, H and Probhu, S. (2009) Sutures and Source of funding suturing techniques in skin closure. Indian J. Dermatol. Vener. Leprol. 75, 425-434. None. Love, C.L., Garcia, M.D., Riera, F.R and Kenney, R.M. (1991) Evaluation of measures taken by ultrasonography and caliper to estimate testicular volume and predict daily sperm output in the stallion. J. Acknowledgements Reprod. Fertil. Suppl. 44, 99-105. Dr Fayaz Paala, Department of Animal Husbandry, Drung, Malmgren, L., Andresen, O. and Dalin, A.M. (2001) Effect of GnRH Badgam, Jammu and Kashmir State, India, extended his immunisation on hormonal levels, sexual behaviour, semen quality and testicular morphology in mature stallions. Equine Vet. J. 33, 75- unconditional support. 83. Mason, B.J., Newton, J.R., Payne, R.J. and Pilsworth, R.C. (2005) Costs Authorship and complications of equine castration: a UK practice-based study comparing standing non-sutured and recumbent sutured M.Y. Mir contributed to study execution and preparation of techniques. Equine Vet. J. 37, 468-472. the manuscript. M.R. Fazili contributed to study design, study Mathew, N.S. and Hartsfield, S.M. (1993) Using injectable anaesthetic execution, data interpretation and preparation of the drugs safely in horses. Equine Pract. 88, 154-159. manuscript. K.H. Dur contributed to anaesthesia Okwee-Acai, J., Acon, J., Okello-Owiny, D., Agwai, B. and Oloya, J. maintenance. M.S. Mir and S. Qureshi contributed to data (2008) Evaluation of pinhole castration as an alternative analysis and interpretation. All authors gave their final technique for goat sterilization. Bull. Anim. Health Prod. Afr. 56, 299-306. approval of the manuscript. Ponvijay, K.S. (2007) Pinhole castration, a novel minimally invasive technique for in situ spermatic cord ligation. Vet. Surg. 36, 74-79. References Ragle, C.A., Southwood, L.L. and Schneider, R.K. (1998) Injury to abdominal wall vessels during laparoscopy in three horses. J. Am. Abid, T.A. and Baghdady, E.F. (2013) Evaluation of pinhole castration Vet. Med. Assoc. 212, 87-89. technique in Iraqi local breed bucks: morphological and histological assessment. J. Anim. Vet. Adv. 12, 423-427. Saifzadeh, S., Hobbenaghi, R., Asri-Rezaei, S., Shokouhi, F.S.J., Dalir- Naghadeh, B.I. and Rohi, M.S.I. (2008) Evaluation of ‘Section- Abu-Ahmed, H.M., El-Kammar, M.H., EL-Neweshy, M.S and Abdel- Ligation-Release (SLR)’ technique devised for castration in the Wahed, R.E. (2012) Comparative evaluation of three in-situ stallion. Reprod. Domes. Anim. 43, 678-684. castration techniques for sterilizing donkeys. Incision-ligation (a novel technique), section-ligation release, and pinhole. J. Equine. Schumacher, J. (1992) Surgical disorders of the testicle and associated Vet. Sci. 32: 711-718. structures. In: Equine Surgery, 1st edn., Ed: J.A. Auer, Saunders, Philadelphia. pp 674-703. Baba, M.A., Fazili, M.R., Athar, H., Mir, M.S., Moulvi, B.A. and Khan, I. (2013) Pinhole castration technique, an alternative to orchiectomy Schumacher, J. (1996) Complications of castration. Equine Vet. Educ. in stray dogs. Anim. Reprod. Sci. 137, 113-118. 8, 254-259. Badawy, A.M. (2009) Percutaneous ligation of spermatic cord as an Schumacher, J. (2012) Testis. In: Equine Surgery, 4th edn., Ed: J.A. Auer alternative to opened castration in donkeys. Benha Vet. Med. J. and J.I. Stick, Elsevier, St. Louis. pp 804-836. 20, 24-41. Searle, D., Dart, A.J., Dart, C.M. and Hodgson, D.R. (1999) Equine Boothe, H.W. (1985) Suture materials and tissue adhesives. In: Textbook castration: review of anatomy, approaches, techniques and of Small Animal Surgery, 1st edn., Ed: D.H. Slatter, Vol.1. W.B. complications in normal, cryptorchid and monorchid horses. Aust. Saunders, Philadelphia. pp 235-244. Vet. J. 77, 428. Desmaizieres, L.M., Martinot, S., Lepage, O.M., Bareiss, E. and Cadore, Tamadon, A., Nikahval, B., Sepehrimanesh, M., Mansourian, M., fi J.L. (2003) Complications associated with cannula insertion Naeini, A.T. and Nazi , S. (2010) Epididymis ligation: a minimally

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invasive technique for preparation of teaser rams. Vet. Surg. 39, Voermans, M., Rijkenhuizen, A.B.M. and Van-Der-Velden, M.A. 121-127. (2006) The complex blood supply to the equine testis as a Trotter, G.W. (1988) Normal and cryptorchid castration. Vet. Clin. N. cause of failure in laparoscopic castration. Equine Vet. J. 38, 35-39. Am. Equine Pract. 4, 493-513. Williams, D. (1998) Catgut sutures: an exercise in discretion. Med. Van-Der-Velden, M.A. and Rutgers, L.J.E. (1990) Visceral prolapse after Device Technol. 9, 6-8. castration in the horse, a review of 18 cases. Equine Vet. J. 22, 9-12.

Review Article How to critically appraise a paper G. L. Pinchbeck* and D. C. Archer Department of Veterinary Epidemiology and Population Health, Institute of Infection and Global Health and Institute of Veterinary Science, Faculty of Health and Life Sciences, University of Liverpool, Leahurst Campus, Neston CH64 7TE, UK *Corresponding author email: [email protected]

Keywords: horse; critical appraisal; study design; bias; confounding; validity; evidence-based veterinary medicine

Summary mastered to practice evidence-based medicine. The Royal Critical appraisal skills enabling assessment of the validity College of Veterinary Surgeons recognises “How to evaluate and importance of evidence are essential for clinicians to evidence” as an essential day one competence required of make informed decisions regarding what new information all veterinary surgeons (RCVS) and “critical analysis of new should be incorporated into their clinical practice. This review information and research findings relevant to veterinary highlights key points to consider in a critical review medicine” is a core competency for accreditation by the concentrating on common study designs used in the equine American Veterinary Medical Association (Anon AVMA). literature. These requirements recognise the essential role of critical appraisal for clinicians’ decision-making regarding what new information, among the vast array available, should be Introduction incorporated into their clinical practice. A vast number of veterinary papers are published every year in a variety of journals. Although many of these are in Critical appraisal reputable journals, and have been through a process of peer-review by experts, the quality may still be variable Critical appraisal is a formal, unbiased, systematic approach to (Pocock et al. 2004). In addition, there are many sources of assessing the quality and relevance of evidence presented in nonpeer-reviewed literature, including textbooks, reports and a paper and its applicability to decision making for our proceedings and the internet. Formal systematic reviews are patients. It includes evaluation of the appropriateness of the beginning to appear in the equine veterinary literature (Allen study design for the research question, and a careful et al. 2012; Sullivan et al. 2015; Dominguez et al. 2016), and assessment of whether the study conforms to specific criteria, RCVS Knowledge and the Equine Veterinary Education are related to the study design. It should be a balanced beginning to publish knowledge summaries (or critically assessment of benefits and strengths of research against its appraised topics) (RCVS Knowledge 2017). However, it is flaws and weaknesses. The assessment of methodological usually up to the individual reader to assess the scientific quality should be done without consideration (or even validity, strength of evidence and practical relevance of knowledge) of the results, so as to avoid interpretation bias results presented in a paper and the extent to which they (Kaptchuk 2003), which arises because interpretation of can be applied to the particular question they are interested findings is rarely completely independent of our previous beliefs in. To assist the process of critical appraisal a number of or preconceptions. For example, higher standards of evidence veterinary and non-veterinary organisations have online may be required in the case of study findings that contradict resources and checklists that can be used as aids (CASP an individual’s initial expectations, compared with a study that 2017; CEBM 2017; CEVM 2017; EBVMA 2017). More detailed agrees with these expectations (confirmation bias). checklists exist for examination of different specific study For the equine clinician with limited epidemiological or types such as randomised controlled trials (RCT), validating statistical knowledge, it can be difficult to critically appraise diagnostic or screening tests, qualitative studies and the study design, statistical analyses used and whether the systematic review or meta-analyses (Greenhalgh 2001c; CASP conclusions drawn can be justified based on the material checklist 2017; CEBM 2017). In addition, a number of presented by the authors. The following sections highlight key reporting guidelines have also been developed for various points that should be considered in a critical review (Table 1), study designs (More 2010), many of which can be found on concentrating on randomised controlled trials and the EQUATOR network website (www.equator-network.org). observational studies, which are the most common study Specific guidelines, with checklists, widely in use include those designs used in the equine literature. for RCTs (CONSORT) and for strengthening the reporting of observational studies (STROBE) (von Elm et al. 2008) available What is the main purpose of the study? at https://www.strobe-statement.org/index.php?id=available- This includes the clinical question the study seeks to address checklists. and what hypothesis is being tested. Not all research studies Evidence-based medicine, defined by Sackett et al. aim to test a single definitive hypothesis and qualitative (1996) as “the conscientious, explicit and judicious use of research studies investigate particular issues in a broad, current best evidence in making decisions about the care of open-ended way (Christley and Perkins 2010). The relevance individual patients”, is much more than just critically assessing of the study question and whether it measures an outcome papers. However, critical appraisal is a key skill that must be that is relevant to your clinical practice, e.g. return to athletic

© 2018 The Authors. Equine Veterinary Education published by John Wiley & Sons Ltd on behalf of EVJ Ltd This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. EQUINE VETERINARY EDUCATION / AE / FEBRUARY 2020 105

TABLE 1: Checklist to aid critical appraisal of a paper 1. What is the main purpose What was the main aim/hypothesis of the study? of the study What was the exposure or intervention? What was the outcome and how was it measured? What were the main results? What was the study population? What, where, when, inclusion/exclusion criteria?

2. What type of study What was the study design and was this appropriate? design was used

3. Is the study internally Bias and Are the results likely to be affected by selection/sampling bias? valid? confounding Are the results likely to be affected by observation/measurement bias? If an intervention trial, were the assessments of outcomes blinded? Are the results likely to be affected by confounding? If a clinical trial, was the allocation adequately randomised?

Statistical Is there sufﬁcient statistical power? methods Are appropriate statistical methods used and results interpreted appropriately?

Causality Can a temporal relationship be ascertained? Is the relationship important/strong? Is there a dose–response relationship? Can the results be explained by noncausal explanations?

4. External validity Can the study results be applied more widely to other populations and to the population under your care? Were all clinically important outcomes considered? Are the outcomes assessed of relevance to your patients?

5. Other factors Who are the authors and is there any potential for conﬂict of interest? Are the results consistent with other evidence? Did the study have appropriate ethical approval?

function or reduced mortality, and whether it adds anything more common in the future as a greater number of high new to the literature is also important to consider. quality primary clinical research studies (particularly Most research studies will evaluate one of the following randomised controlled trials) are reported. Primary clinical (Greenhalgh 2001a): Therapy (efﬁcacy of a drug treatment, research studies can be experimental or observational. In surgical procedure or other intervention); Causation (if a experimental studies, such as the RCT, the investigator suspected risk factor is related to development of a particular controls the allocation of the intervention (e.g. a new disease); Prognosis (outcome of a disease following treatment vs. no treatment or an existing treatment) to a treatment/diagnosis); Diagnosis (the validity and reliability of randomly selected subset of the study subjects and then a new diagnostic test and superiority to any existing tests) or compares between groups of study animals to make Screening (tests applied to a population to detect disease). inferences about the effect of the intervention on the outcome of interest. The RCT can provide strong evidence What type of study design was used (and is this the and is often regarded as the highest standard of evidence to most appropriate for the question addressed)? guide clinical decision-making due to key aspects of design Understanding the type of study that has been performed is that aim to avoid bias. Although still infrequent in equine a prerequisite to evaluation of the strength of evidence clinical research, RCTs are becoming more common (Sabate provided by the study. In addition, knowledge of the design et al. 2009; Talbot et al. 2013; Higler et al. 2014). The will enable the reader to determine if the study in question observational studies (cohort, case control and has been appropriately designed and conducted (assessing cross-sectional) are next in the hierarchy, and these are the internal validity), and, if not, whether this should decrease relatively frequently used in the equine research literature. the strength of belief in the results. Sometimes, authors will Descriptive studies such as case reports and case series are state that they have performed a particular study design but very common in the equine research literature. However, careful reading of the methods may contradict this. these are not designed to test an association between a The hierarchy of evidence (Fig 1), which ranks the relative therapy and a treatment (Grimes and Schulz 2002). If an strength of evidence carried by the different types of study author draws conclusions about the merits of a particular when making decisions about clinical interventions, is well therapy from a case report or series, this should be regarded recognised. The pinnacle of the hierarchy is reserved for as the author’s opinion/conjecture only, as these provide papers in which all the primary studies on a clinical question limited evidence to support this. or subject are critically appraised according to rigorous criteria (the systematic review) and meta-analysis, which Is the study internally valid? integrates the numerical data from more than one study. The Internal validity is concerned with the quality of the study as it latter studies are relatively infrequent in equine veterinary applies to the population that is being studied, and its research (Calzetta et al. 2017), but are likely to become assessment involves asking the question: did the researchers

SR & MA

Randomised controlled trial

Cohort studies

Case control studies

Cross sectional studies

Case reports and case series

Editorials and opinions

In vitro research

Fig 1: Hierarchy of evidence. SR & MA = Systematic reviews and meta-analyses. Adapted from: SUNY Downstate Medical Center. Medical Research Library of Brooklyn. Evidence Based Medicine Course. A Guide to Research Methods: The Evidence Pyramid: http:// library.downstate.edu/EBM2/2100.htm]. do things properly? Once you have established that the racing is biased towards a healthy horse population) and paper addresses a relevant clinical question, and that an detection bias (controls wrongly classified when they have appropriate study design has been used, the methodology the disease of interest because, for example, they did not should be critically appraised for quality and the strength of receive the same examination protocol as cases). evidence of the work presented. This should consider whether • Information bias: Occurs when the outcomes, exposures of potential sources of bias or confounding were addressed, interest (factors measured) or other data are incorrectly whether the study was performed according to the original classified or measured. This might be, for example, due to protocol and if appropriate statistical methods were use of poor diagnostic criteria or tests, or differing performed correctly. In some instances, the reader may be application of data collection techniques or tests presented with insufficient information to appraise the depending on the outcome or exposure status of the reliability and such papers should be interpreted with caution. subject (for example, administering a questionnaire face-to-face for cases but by post for controls) Bias and confounding • Confounding bias: This is the mixing of the effects of two or Many of the aspects of design which need to be considered more factors. We might think that we are measuring the to determine internal validity of a study are concerned with association between an exposure factor and an outcome, assessing whether systematic bias has been avoided or but the association we observe actually includes the minimised throughout the study. Bias can be defined as the effect of one or more other variables. Hence, our systematic (nonrandom) error in design, conduct or analysis assessment of the association between the exposure of a study resulting in mistaken estimates, and different study factor and the outcome is biased (or confounded). For designs require different steps to reduce bias (see next example, if we wished to assess whether transport, as an section). Bias can occur due to the way populations are exposure factor, was associated with the risk of colic, we sampled, data are collected, or are analysed. Unlike random would need to take into account the effects of one or error, increasing the sample size will not decrease systematic more potential confounding factors (e.g. change of bias. There are numerous types of bias (Sackett 1979; Dohoo forage or change in time at pasture), that are et al. 2009) but these can be considered under three hypothesised risk factors for colic but are also likely to be headings: associated with transport. To avoid bias and get an • Selection bias: This bias occurs when the composition of accurate estimate of the effect of transport on colic, these study subjects or participants in a research project confounding factors must be taken into account. Once systematically differs from the source population. There are you have identified the study type and considered the many different sub-types of this type of bias including exposures and outcome factors, you can make a list of choice of comparison groups, nonresponse bias (e.g. possible confounders and then examine the paper to see respondents differ compared with those who do not how the authors have dealt with these (if at all). respond in a questionnaire study), follow-up bias (loss to follow-up is different between groups being compared), Particular types of bias may be more likely with specific selective entry bias (e.g. use of horses that are currently study designs, as outlined in the next section. Specific points

that should be considered in the appraisal of these studies are bias due to loss to follow-up. Where potential confounding also summarised (adapted from Young and Solomon 2009): factors are identified prior to prospective cohort studies, these can be measured and taken into account in the Randomised controlled trials (RCTs). The RCT is a prospective analysis, but in retrospective studies this may be difficult or study designed to assess the effect of one (or more) impossible if these factors were not measured or were treatments or interventions compared with a control group measured poorly or inconsistently. Your checklist for these that may consist of no treatment, a placebo or a studies should consider: comparator treatment (for example, a current standard therapy). Key design aspects that provide the best means of • Is the study prospective or retrospective? avoiding bias include the process of random allocation to • Is the cohort representative of a defined group/ treatment groups, which aims to ensure that treatment population? groups are equivalent in terms of both known and unknown • Were all important confounding factors identified, confounding factors and hence any differences in outcomes adequately measured and adjusted for in the design or can therefore be ascribed to the effect of treatment. The analysis? process of blinding in RCTs, whereby participants and those • Were all important exposures and outcomes measured who are assessing the outcomes (and in some cases also accurately, objectively and equivalently in all the cohort those analysing the data) are unaware of intervention subjects (including irrespective of exposure status)? assignment, reduces information or measurement bias. The • Were there any losses to follow-up and could this bias the CONSORT guidelines (Anon 2017) provide a comprehensive findings? checklist specifically for RCTs but specific points for consideration should include: Case–control studies. Case–control studies are common in • Was the process of treatment allocation properly the equine literature and are ideal for investigation of risk described and truly random? factors where the outcome of interest (e.g. a specific disease) • Were the groups comparable in all important aspects is rare and it would be impractical to perform a prospective except for the variable being studied? cohort study (e.g. too many horses needed/would take too • Were participants and researchers ‘blinded’ to long to perform). In case–control studies, cases are only participants’ treatment group? recruited once they have developed the outcome of interest • Were primary and secondary outcome measures, properly and exposure data are then evaluated to determine whether defined and objectively assessed? exposures of interest differ between the cases and controls. • Were all participants who were randomly allocated a Appropriate control selection is often the most difficult aspect treatment accounted for in the final analysis? of a case–control study as controls must come from the same population of cases to avoid bias. Controls should be subjects Papers describing comparative studies in which subjects who would have entered the study and become cases if the are allocated to intervention or control groups in a outcome had occurred. Exposure data are often collected nonrandom manner are not randomised trials, and can be by questionnaire or from existing records. Recall bias is termed “other controlled clinical trials” (Greenhalgh 2001a). another potential issue in these types of studies where owners/ There is a high risk of bias and confounding in these types of carers cannot remember exact details about historical studies. For example, if a horse is not randomised to a factors, and is a particular concern where this may differ particular therapy or surgical procedure, a clinician may between groups (e.g. owners of horses who developed the decide upon the therapeutic/surgical treatment of that disease of interest may recall specific factors more accurately patient based on particular characteristics or specific than owners of control horses). In addition, recording of features of the disease, such as severity (leading to selection subjective rather than objective data may result in different bias); hence, any difference in outcome between the groups responses from case or control owners leading to bias. may relate to biased selection (grouping) of the patients Specific points to consider in these studies are therefore: themselves rather than the therapy/surgical intervention performed. If the paper you are looking at is a • Were cases clearly defined and eligibility and nonrandomised controlled clinical trial, you must use your ascertainment described? judgement to decide if the baseline differences between the • Were controls appropriately selected and drawn from the groups are likely to have been so great as to undermine same population as the cases, using the same eligibility claims regarding any differences (or lack of differences) criteria? ascribed to the intervention. • Were exposures measured equivalently for cases and controls? Cohort studies. Cohort studies involve the follow-up of study • Is recall bias likely? participants with varying exposures forward, to observe • Were all important confounding factors identified and which animals develop the outcome(s) of interest (e.g. adjusted for in design (for example by matching) or clinical disease or mortality), and to determine the effect of analysis? exposures on the outcome. These studies are commonly prospective, where data are specifically collected for the purposes of the study, but may also be retrospective in Cross-sectional studies. These studies provide a ‘snapshot’ in nature, using data that have been routinely collected for time to determine information (for example, disease another purpose e.g. clinical or race records. There is a prevalence) about a population of interest. Concurrent potential for confounding in these types of studies and for measurement of exposures of interest may also be used to

elucidate factors associated with the outcome. Specific Strength of evidence and causality aspects of study design that should be considered include: Other factors that should be considered include an interpretation of whether the results infer causality (Hill 1965): • Were the eligibility criteria and methods of selection of the study sample clearly defined? • Temporal relationship – does the study demonstrate that • Was a representative sample obtained (e.g. sufficiently the exposure/intervention preceded the disease/outcome high response rate) of interest? It is often not possible to demonstrate this in • Were all relevant exposures, potential confounders and cross-sectional or case–control studies. outcomes measured accurately? • Strength of association – Is the relationship sufficiently • Is it certain that the exposure of interest occurred prior to ‘strong’ to be clinically/biologically important? This can development of the outcome? usually be evaluated by looking at the size of ratio measures (e.g. odds/risk ratio or differences in risk/rate of disease) and the precision of the estimate of effect. Statistical methodology Whether the effect would still be sufficiently strong at either Although it is unlikely that most readers will be able to end of the 95% CI should be considered. critically evaluate every type of statistical analysis that may • Dose response – does the risk of disease or outcome of be used in clinical studies, it is still important to be able to interest increase with increasing levels of the exposure? critique key issues. If the statistical tests in the paper are The most commonly cited example of this is in human uncommon, the authors should provide justification why they medicine and the fact that the death rate from cancer of have used them and describe them in detail or include a the lung rises linearly with the number of cigarettes smoked relevant reference. daily. Demonstrating a strong dose–response relationship For many studies, and in particular RCTs, a sample size adds a great deal to the simpler evidence that an calculation is a crucial prerequisite. The sample size should be exposure increases the risk of disease. clearly defined and justified using appropriate calculations • Noncausal explanations – does the association between and should be big enough to have a high chance of an exposure and outcome of interest make biologically detecting, as statistically significant, a worthwhile effect if it and epidemiological sense (and is it consistent with other exists. If ultimately the study did not then reach the described studies or what is known about disease mechanisms) or sample size, then it may be underpowered and lead to could this relationship be due to something else? erroneous conclusions. The statistical methods used for any comparisons of the data should be clearly stated and any key assumptions, and How externally valid is the study and is it applicable to evidence that these have been met, should be described patients under your care? (e.g. assumptions of normal distributions, multiple testing). Once you have determined a study is of good quality (i.e. Statistical tests are either parametric (i.e. they assume that internally valid), the next step is to evaluate if the study has the data were sampled from a normal distribution) or external validity. External validity relates to how well the results nonparametric (i.e. they do not assume that the data were can be generalised to other populations and in particular your sampled from any particular distribution). Inappropriate target population, i.e. patients under your care. The horses statistical tests commonly used include use of parametric studied may differ from those under your care if, for example, tests when data are not normally distributed, conducting they had more or less co-morbidities; they were of different multiple testing and ignoring clustered data. Greenhalgh breed or under very different management routines. In (2001b) provides further details, including some frequently addition, you should consider if the outcomes assessed are of used statistical ‘tricks’ you should be aware of. relevance to your patients, or if clinically relevant outcomes The results section should include sufficient description of have been ignored. Remember, however, a study can only the data to enable readers to understand how the authors have external validity if it is also internally valid. arrived at their conclusions. This usually entails provision of point estimates of effect (or difference) and measures of variation Other factors to consider (usually the 95% confidence interval). The Equine Veterinary The findings of the study should then be considered together Journal provides useful guidelines and a statistical checklist with other evidence to determine whether they are (Christley 2015) but specific points to consider include: consistent with other studies or whether the findings contradict previous studies. It is also important to consider • Are groups comparable and, if necessary, adjusted for whether the authors have any potential conflicts of interest baseline differences? and, if so, whether these have been recognised. For • Have the data been analysed according to the original example, commercial funding of a study might lead to a protocol? conflict of interest (e.g. if there is potential for increased sales • Are statistical tests appropriate for the types of data of a commercial product based on study findings, any described? negative findings may not be presented). If potential conflicts • If the statistical tests in the paper are uncommon, why of interest exist this should be addressed e.g. for commercially have the authors chosen to use them? funded research there should be a statement about whether • Have confidence intervals been calculated and do the the study design, analysis and interpretation of results was authors’ conclusions reflect these? performed independently of the funding body. In addition, • Have assumptions been made about the nature and the reader should consider whether the study has been direction of causality? Remember statistical association performed with appropriate ethical approval (Bertone 2013), does not provide direct evidence of causation. including informed owner consent, although ethical

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guidelines and standards (including for reporting) do vary Dominguez, M., Munstermann, S., De Guindos, I. and Timoney, P. (2016) between different countries and journals, and in some Equine disease events resulting from international horse movements: systematic review and lessons learned. Equine Vet. J. 48, 641-653. settings local institutional ethics committees may not exist. von Elm, E., Altman, D.G., Egger, M., Pocock, S.J., Gøtzsche, P.C. and Vandenbroucke, J.P. (2008) STROBE Initiative. The Strengthening Conclusion the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. “http:// Ultimately, it is often up to the reader to determine the extent download.journals.elsevierhealth.com/pdfs/journals/0895-4356/PIIS0 to which s/he feels the results presented in a paper are reliable 895435607004362.pdf” \t “_blank” \o “Opens external link in new and if, and how the findings may potentially benefit patients window" J. Clin. Epidemiol. 61, 344-349. under their care. There is no single tool that can be used to Equine Vet. J. Clinical Evidence in Equine Practice online collection. perform critical appraisal of all types of study, however there Edited By: C. M. Marr. http://onlinelibrary.wiley.com/journal/10. 1001/(ISSN)2042-3306/homepage/clinical_evidence_in_equine_ are numerous online resources and checklists that can be used practice_online_collection.htm Accessed: 17/8/2017 as an aid. The critical appraisal checklist (Table 1) is not Evidence Based Veterinary Medicine Association (2017) https:// exhaustive but can be used as a guide to identify whether the ebvma.org/continuing-education/ Accessed: 10/8/2017. research documented in a paper conforms to certain criteria Greenhalgh, T. (2001a) Chapter 3: getting your bearings (what is this that are important in epidemiological studies. paper about?). How to read a paper: the basics of evidence based medicine, 2nd edn., B.M.J. Books, London. pp 39-58. Authors’ declaration of interests Greenhalgh, T. (2001b) Chapter 5: statistics for the non-statistician. How to read a paper: the basics of evidence based medicine, No conflicts of interest have been declared. 2nd edn., B.M.J. Books, London. pp 76-93. Greenhalgh, T. (2001c) Appendix 1: checklists for finding, appraising, and Ethical animal research implementing evidence. How to read a paper: the basics of evidence based medicine, 2nd edn., B.M.J. Books, London. pp 200-209. Not applicable to this review article. Grimes, D.A. and Schulz, K.F. (2002) Descriptive studies: what they can and cannot do. Epidemiology Series. Lancet 359, 145-148. Source of funding Higler, M.H., Brommer, H., L’Ami, J.J., de Grauw, J.C., Nielen, M., van Weeren, P.R., Laverty, S., Barneveld, A. and Back, W. (2014) The None. effects of three-month oral supplementation with a nutraceutical and exercise on the locomotor pattern of aged horses. Equine Authorship Vet. J. 46, 611-617. Hill, A.B. (1965) The environment and disease: association or causation. Both authors contributed to the writing and approved the Proc. R. Soc. Med. 58, 295-300. fi nal manuscript. Kaptchuk, T.J. (2003) Effect of interpretive bias on research evidence. BMJ 326, 1453-1455. References More, S. (2010) Improving the quality of reporting in veterinary journals: how far do we need to go with reporting guidelines? Vet. J. 184, Allen, K.J., Christley, R.M., Birchall, M.A. and Franklin, S.H. (2012) A 249-250. fi systemic review of the ef cacy of interventions for dynamic Pocock, S.J., Collier, T.J., Dandreo, K.J., de Stavola, B.L., Goldman, intermittent dorsal displacement of the soft palate. Equine Vet. J. M.B., Kalish, L.A., Kasten, L.E. and McCormack, V.A. (2004) Issues in 44, 259-266. the reporting of epidemiological studies: a survey of recent American Veterinary Medical Association. https://www.avma.org/ practice. BMJ 329, 883-887. 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Critically Appraised Topic Can ocular administration of atropine cause colic? R. Coomer Cotts Equine Hospital, Narberth, Pembrokeshire, UK. Corresponding author email: [email protected]

Introduction and 24 mg. Two of the four resolved without treatment in 2 h; the other two received symptomatic pain relief, Atropine is a competitive muscarinic antagonist drug in resolving pain in 2 and 6 h respectively. This experimental cholinergic synapses of the parasympathetic nervous system. scenario differs markedly to normal clinical application, e.g. It is applied ocularly for its ability to block contraction of the high dose rates, the lack of ocular disease, pain or other ciliary muscle. It limits pain through ciliary muscle relaxation, systemic drugs. preventing synechia formation in horses. The potential for The pharmacokinetics of ocular atropine has not been systemic absorption, secondary inhibition of parasympathetic measured in horses, though it has in man. Lahdes et al. (1988) gut motility and consequent colic is a perennial concern. instilled 40 microlitres of 1% atropine solution (0.4 mg) to one eye of healthy human volunteers and recorded systemic PICO question plasma levels for 90 min. Levels peaked at 8 min. No effect on heart rate, blood pressure or salivary secretion occurred, In horses with ocular disease, does topical treatment with the main side-effect concerns in human medicine. The atropine increase the risk of colic compared to no topical extrapolated dose rate in this study was 0.005 mg/kg (80 kg atropine treatment? body weight), an order of magnitude lower than Ducharme and Fubini (1983). The bioavailability of ocularly applied Search strategy atropine was measured by Kaila et al. (1999). They supplied 0.3 mg atropine, intravenously or ocularly, measuring Literature searches were conducted for articles published in subsequent plasma concentrations. Mean bioavailability was English on the MEDLINE database (inception to June 2017), the 63.5 Æ 28.6%, range 19–95%. Route of administration did not CAB Abstracts database (1973 to January 2017) and using alter terminal half-life, whilst peak plasma concentration also Google Scholar. Search terms included ‘equine’, ‘atropine’, varied widely, range 3–60 min after ocular administration. Two ‘pharmacokinetics’, ‘ocular’, ‘ocular disease’ and ‘colic’. features are noteworthy: (a) bioavailability was so high; (b) Articles were included if they reported either systemic or ocular that so much individual variation in rate and degree of administration of atropine in horses and evidence of subsequent absorption occurred. The authors surmised that some colic. Articles describing single cases or those that did not variation was due to overflow losses in the conjunctiva. contain original data (i.e. expert opinion) were excluded. Atropine also bonds to melanin: heavily pigmented eyes show a slower onset and longer duration of action (Jaanus Quality of evidence et al. 1989). The searches identified 11 papers deemed relevant. The main fi What about horses with a painful eye treated ndings are shown in Table 1. A series of experimental studies with atropine? looking at the effects of intravenous atropine on intestinal activity and colic dating from the 1980s are detailed in Within their review paper, McLaughlin et al. (1991) recorded Table 1. They show fairly convincingly that mild to moderate a series of 15 horses hospitalised 5–14 days for ocular disease. self-resolving colic can be induced by i.v. atropine. Given All received topical atropine at concentrations from 0.25 to that it is a route not used for ophthalmic disease, these 1%, from twice daily to continuous lavage. No reduced papers are not discussed further here. intestinal motility or colic signs were observed. Patipa et al. (2012) published a retrospective review Can topical ocular atropine cause colic? investigating risk factors for colic in 337 equids hospitalised for ocular disease. The authors recognised that such horses were Zekas et al. (1998) published results obtained by treating exposed to many known risk factors for colic, e.g. transport, four healthy horses with topical ophthalmic atropine every 4 management changes, pain, general anaesthesia, h for 8 treatments, using a crossover design with saline prolonged systemic and topical drug treatment. Overall, 72 control. Intestinal myoelectric activity was measured using of 337 horses colicked, nine needing surgery. Five colic pre-implanted electrodes, as well as calculating faecal fatalities occurred. Results of univariable analysis identified transit time using plastic beads and clinical assessment for three significant colic risk factors: age (horses up to one or colic. Intestinal activity was suppressed in all horses and one over 21 years), longer hospitalisation time, use of atropine. had increased transit time. Two of four horses colicked after Atropine became nonsignificant in the final multivariable 16 and 32 h, respectively. Williams et al. (2000) supplied six analysis (P = 0.52, OR 1.29; 95% CI 0.61–2.7). horses with 1 mg topical atropine solution per hour for up to Perhaps recognising that the rate of colic reported by 24 h, or until signs of colic developed. All horses lost audible Patipa et al. (2012) was unusually high, Scherrer et al. (2016) borborygmi for up to 18 h. Four colicked, after 11, 14, 22 retrospectively compared 105 horses hospitalised for ocular

TABLE 1: Summary of the papers deemed relevant in answering the CAT: in horses with ocular disease, does topical treatment with atropine increase the risk of colic compared to no topical atropine treatment?

Relevant papers

First author, year Patient group Study type Outcomes Key results Study weaknesses

Ducharme 10 ponies supplied i.v. Experimental Inappetence <2 h and Colic duration 1– Intravenous not and Fubini atropine at 2 dose cohort study 7.5 h. 3/10 ponies 4 h, all resolved ophthalmic route, (1983) rates colicked: 1/5 lower without healthy ponies, dose, 2/5 at higher. treatment. borborygmi used to assess motility Adams 4 horses, i.v. atropine, Experimental Intestinal activity No colic occurred Intravenous not et al. (1984) motility measured by cohort study suppressed from 65 ophthalmic route, intestinal electrodes (control) to 315 min healthy horses Erhardt and 6 horses, i.v. atropine, Experimental Intestinal activity No colic occurred Intravenous not Lowe (1987) high ﬁdelity cohort study suppressed for 1 h ophthalmic route, recording used to healthy horses, assess motility recording only 1 h Zekas 4 horses, ophthalmic Experimental case Decreased Colic occurred Healthy horses, no et al. (1998) atropine 2 mg q. control crossover myoelectric activity after 4th and 8th ophthalmic disease 4h9 40 h, with study in all, 2/4 colicked topical dose in 2 crossover saline (16 and 32 h), 1/4 of 4 horses control. Motility increased faecal measured by transit time intestinal electrodes and faecal transit time Williams 6 horses, ophthalmic Experimental case Decreased Colic occurred Healthy horses, no et al. (2000) atropine 1 mg q. control crossover borborygmi, 4/6 after 11th, 18th, ophthalmic disease, 1h9 24 h, with study horses developed 21st and 22nd higher doses than crossover colic, all resolved doses in 4 of 6 recommended in methylcellulose spontaneously horses ophthalmic disease control Lahdes 8 human volunteers Prospective case Levels peaked at No effect on heart Humans not horses, et al. (1988) undergoing ocular control study 860 Æ 402 pg/mL at rate, blood anticholinergic side surgery, ophthalmic 8 min pressure or effects of concern are atropine 0.4 mg with salivary secretion different plasma occurred measurement Kaila 6 human volunteers Randomised case Mean bioavailability Bioavailability, Humans not horses, et al. (1999) given 0.3 mg i.v. or control crossover 63.5 Æ 28.6%, range peak plasma anticholinergic side ophthalmic atropine study 19–95%. Terminal conc and half life effects of concern are with plasma half-life 1.4–4.3 h all very variable different measurement regardless of route. Peak plasma conc. 3 –60 min after ocular McLaughlin 15 horses hospitalised Retrospective No apparent No colic occurred Small case number, no et al. (1991) for clinical cohort study, reduction in GI analysis, statistics or ophthalmic disease, described within motility, reduced control group all received topical larger review faecal output or atropine paper colic Patipa 337 horses hospitalised Retrospective 72 of 337 horses Atropine non- No control group, high et al. (2012) for clinical cohort study (21.4%) colicked, 9 signiﬁcant in rate of background ophthalmic disease, needing surgery, 5 multivariable colic suggests some received fatalities occurred as analysis, P = 0.52, management issue, no topical atropine, no a result of colic. OR 1.29; 95% CI details on use of dose or frequency Colic risk factors age 0.61–2.7 atropine presented recorded (<1or>21 years), longer hospitalisation time Scherrer 105 horses hospitalised Retrospective case 8/105 (8%) ocular Ophthalmic Management of et al. (2016) for clinical control study cases and 9/197 (5%) atropine 61/105 orthopaedic different to ophthalmic disease orthopaedic cases (58%), no ophthalmic cases, vs. 196 for colicked, P = 0.36. Of association with retrospective orthopaedics, 17 colics, 1 needed colic, P = 0.97 atropine max twice/ surgery day

and 197 for orthopaedic disease. Just 8/105 (8%) ocular cases References and 9/197 (5%) orthopaedic cases colicked, P = 0.36. Of the Adams, S.B., Lamar, C.H. and Masty, J. (1984) Motility of the distal 17 with colic, one needed surgery. Atropine was administered portion of the jejunum and pelvic flexure in ponies: effects of six to 61/105 (58%) of horses with ocular disease and there was drugs. Am. J. Vet. Res. 45, 795-799. = no association with the risk of colic, P 0.97. The authors Ducharme, N.G. and Fubini, S.L. (1983) Gastrointestinal complications noted that atropine was supplied at a maximum frequency associated with the use of atropine in horses. J. Am. Vet. Med. of twice daily. Assoc. 182, 229-231. Erhardt, E.E. and Lowe, J.E. (1987) Lengthy electronic auscultation of the equine abdomen to monitor the effects of analgesics, sedatives Conclusions and other drugs. Proc. Am. Ass. Equine Practnrs. 33, 525-538. It is possible to induce mild reversible symptoms of colic Jaanus, S.D., Pagano, V.T. and Bartlett, J.D. (1989) Drugs affecting the autonomic nervous system, In: Clinical Ocular Pharmacology, 2nd through frequent administration of topical ocular atropine in edn., Ed: J.D. Bartlett, Butterworth Heinemann, Boston. pp 128-131. normal healthy horses. Systemic bioavailabilty is potentially Kaila, T., Korte, J.M. and Saari, K.M. (1999) Systemic bioavailability of both high and rapid, even if individual horses vary naturally. ocularly applied 1% atropine eyedrops. Acta Ophthalmol. Scand. Lower overall doses, conjunctival overflow losses, variation in 77, 193-196. pharmacokinetics and the presence of more potent risk Lahdes, K., Kaila, T., Huupponen, R., Salminen, L. and Lisalo, E. (1988) factors probably explain why no retrospective clinical studies Systemic absorption of topically applied ocular atropine. Clin. demonstrate an association between ocular atropine and Pharmacol. Ther. 44, 310-314. colic in horses with ocular disease. Therefore, the use of McLaughlin, S.A., Whitley, R.D. and Gilger, B.C. (1991) Ophthalmic ocular atropine at recommended dose rates for clinical atropine in horses: is colic a serious problem?. Equine Vet. Educ. 3, ocular disease does not increase risk of colic. 94-96. Patipa, L.A., Sherlock, C.E., Witte, S.H., Pirie, G.D., Berghaus, R.D. and Peroni, J.F. (2012) Risk factors for colic in equids hospitalized for Author’s declaration of interests ocular disease. J. Am. Vet. Med. Assoc. 240, 1488-1493. Scherrer, N.M., Lassaline, M., Richardson, D.W. and Stefanovski, D. No conflicts of interest have been declared. (2016) Interval prevalence of and factors associated with colic in horses hospitalized for ocular or orthopaedic disease. J. Am. Vet. Ethical animal research Med. Assoc. 249, 90-95. Williams, M.M., Spiess, B.M., Pascoe, P.J. and O’Grady, M. (2000) Not applicable. Systemic effects of topical and subconjunctival ophthalmic atropine in the horse. Vet. Ophthalmol. 3, 193-199. Zekas, L.J., Lester, G. and Brooks, D.E. (1998) The effect of ophthalmic Source of funding atropine on intestinal transit and myoelectric activity in normal adult horses. In: Proc. ACVO, Seattle, WA. pp 21-24. None.

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