ANHIDROSIS IN HORSES IN FLORIDA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0224736
• Project Start Date: Mar 1, 2010
• Project End Date: Feb 28, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
College of Veterinary Medicine

Non Technical Summary
Anhidrosis, or the failure to sweat appropriately, is a common syndrome of exercising horses, especially those in hot and humid climates such as that found in Florida. Anhidrosis usually ends a horse's athletic career. Unfortunately, for such an important disease, very little is known about the cause of anhidrosis and attempts to diagnose, treat or prevent it are little more than witchcraft. This project will attempt to fill in some of the blanks regarding anhidrosis in Florida. (1) A study will be performed to document how commonly anhidrosis occurs in different parts of Florida. We expect that this study will also reveal risk factors that may increase the likelihood of a horse developing the condition. (2) An accurate test will be developed to measure the ability of anhidrotic and normal horses to sweat. This will help accurately diagnose the disease. (3) Skin samples will be taken from normal and anhidrotic horses and these will be examined microscopically using special staining techniques in order possibly to reveal the cause of anhidrosis. (4) We will attempt to develop genetic markers for anhidrosis in horses. If this is successful, it will provide a key indicator of susceptibility to anhidrosis BEFORE the condition develops and point to rational preventative and treatment options.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1020	30%
311	3810	1170	30%
311	3810	1040	20%
311	3810	1090	20%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1020 - Physiology; 1170 - Epidemiology;

Keywords

anhidrosis

sweating

horse

genetic

aquaporin

epidemiologic

Goals / Objectives
The ultimate goal of this study is to understand anhidrosis in horses so that it can be treated and prevented. To meet this goal, I will pursue the following objectives: (1) to survey and describe anhidrosis in the state of Florida; (2) to use epidemiologic techniques to identify risk factors for anhidrosis in the state of Florida; (3) to develop a quantitative intradermal sweat test to accurately identify horses with anhidrosis; (4) to investigate expression of the water channel protein aquaporin 5 in horses with and without anhidrosis; (6) to identify genomic mutations or polymorphisms associated with susceptibility to anhidrosis; (7) develop genetic tests for susceptibility to anhidrosis.

Project Methods
OBJECTIVES 1&2. At least 280 horse premises will be enrolled and surveyed via questionnaire for farm level and horse level risk factors. Among farms with horses affected with anhidrosis, the questionnaire will be structured to gather information on every anhidrotic horse and up to 10 nonanhidrotic horses per farm. The questionnaire will pertain to conditions during the previous calendar year. Both cross-sectional and case control approaches will be used to identify risk factors associated with anhidrosis. OBJECTIVE 3. 6-10 free-sweating horses will be used for the study. Quantitative intradermal terbutaline sweat tests (QITST) will be done monthly for a year. Ambient temperature, relative humidity, and dew-point recordings will be obtained with a portable hygro-thermometer. OBJECTIVE 4. Skin biopsies will be obtained from 5-10 free-sweating and 5-10 anhidrotic horses. Four-micron sections will be incubated with anti-aquaporin 5. Sections of mouse foot pads will be used as positive controls. Sections will be read using a semi-quantitative scoring system for immunostaining and statistical comparisons will be made between aquaporin-5 expression in normal versus anhidrotic horses by Student's t-tests. OBJECTIVE 5. Two different methods will be used to identify disease mutations, first, the candidate gene approach and, if that is not successful, the genetic mapping approach. Candidate gene approach involves identification of a gene(s) likely to contain the causative mutation based on known biochemical function of the gene product or a homologous disease in other species. In this case we will use DNA sequencing of the gene for aquaporin-5 in both normal and affected individuals to determine if a detrimental mutation occurs in the affected individuals. This can be relatively rapidly sequenced for coding sequence mutations by reverse transcriptase PCR from affected and control tissues. Sequencing of candidate genes has been dramatically simplified by the recent publication of the complete equine genome sequence. If this effort is unsuccessful (because aquaporin-5 is not involved), we will use genetic mapping via association analysis of inherited DNA markers to identify the region of the genome containing the disease gene. As a result of the sequencing of the equine genome, there are now tens of thousands of micro-satellite arrays in the genome that can be identified from the genome sequence. Additionally, the Broad Institute of Harvard and MIT's SNP discovery effort in Twilight and seven representatives from both recently developed and ancient breeds has identified 1.5 million single nucleotide polymorphisms in the equine genome. An equine whole genome SNP genotyping chip (~20 SNPs/Mb) is now available, which greatly enhances the power to detect genetic association and find disease genes by analyzing 60,000 SNP markers on a single horse at the same time. We predict that use of these SNP genotyping chips will allow for rapid identification of anhidrotic gene or genes.

Progress 03/01/10 to 02/28/15

Outputs
Target Audience: Horse owners and riders and equine veterinarians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three Merial summer scholars from the DVM curriculum have worked or are working on projects related to these objective that have or will result in refereed publications. One Master's student (Eric Johnson) completed his MS on objective 2 above. Two Residents in Large Animal Medicine (Martha Mallicote and Amy Stieler) in the College of Veterinary Medicine completed their research requirements on projects included in the above objectives and have or will publish first author published reports of the work. How have the results been disseminated to communities of interest? 1. Refereed publications (reported above). 2. Abstracts/lectures at scientific meetings (reported above). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) to survey and describe anhidrosis in the state of Florida. (2) to use epidemiologic techniques to identify risk factors for anhidrosis in the state of Florida OBJECTIVE-To estimate prevalence of and identify factors associated with anhidrosis in horses in Florida. Design-Cross-sectional study and case-control study. ANIMALS-4,620 horses on 500 farms. PROCEDURES-A questionnaire was structured and mailed to farm owners or managers to obtain information related to diagnosis of anhidrosis in horses and exposure factors associated with this condition. The frequency of investigated farm- and animal-level factors was compared between farms and horses affected and not affected with anhidrosis, respectively. RESULTS-The prevalence of anhidrosis was 11% at the farm level and 2% at the animal level. The odds of anhidrosis were 2.13 and 4.40 times as high in farms located in central and southern Florida, respectively, compared with odds for farms in northern Florida. The odds of anhidrosis were 5.26 and 15.40 times as high in show and riding instruction operations, respectively, compared with odds for ranch operations. At the animal level, breed (Thoroughbreds and warmblood horses), foaling place (western or midwestern region of the United States), and family history of anhidrosis were significantly associated with anhidrosis. CONCLUSIONS AND CLINICAL RELEVANCE-This study provides new information on the prevalence of and factors for anhidrosis in horses in Florida. Horses with a family history of anhidrosis should be examined by a veterinarian for diagnosis of this condition before they are exposed to exercise in a hot and humid climate. (3) to develop a quantitative intradermal sweat test to accurately identify horses with anhidrosis The aim of the current study was to quantify sweating responses to intradermal terbutaline in normal horses. Seven Thoroughbred horses were used. Terbutaline (10-fold dilutions from 1000-0.001 mg/l) and a saline control were injected intradermally (0.1 ml/site) and sweat collected for 30 min into absorbent pads taped over each injection site. Tests were performed monthly for 11 successive months and temperature, relative humidity and dewpoint were measured at the time of testing. There was no significant effect (P
Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: MacKay, RJ, Mallicote, M, Hernandez, JA, Craft, WF, Conway, JA. A review of anhidrosis in horses. Equine Vet Educ. 2015, 27:192-199.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Stieler AL, Sanchez LC, Mallicote MF, Martabano BB, MacKay RJ. Macrolide induced hyperthermia in foals. Role of impaired sweat responses. J Vet Intern Med 2014, 28:1109, Abstract E-26.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Johnson EB, MacKay RJ, Hernandez JA. An epidemiologic study of anhidrosis in horses in Florida. 2010, 236:1091-1097.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Stieler AL, Sanchez LC, Mallicote MF, Martabano BB, Burrow JA, and MacKay RJ. Macrolide-induced hyperthermia in foals: Role of impaired sweat responses. Submitted to Equine Veterinary Journal
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: AL Stieler, LC Sanchez, MF Mallicote, AV Muniz, JA Burrow, RJ MacKay. Effect of rifampin on erythromycin-induced anhidrosis in foals. Accepted. J Vet Intern Med
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: AL Stieler, LC Sanchez, MF Mallicote, S Westerterp, JA Burrow, RJ MacKay. A comparison of the effects on sweating of three macrolide antibiotics used in foals. Accepted. J Vet Intern Med
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Mallicote, M.F., Medina, C.I., Xie, H., Zilberschtein, J., Atria, S., Manzie, M., Hernandez, J. and MacKay, R.J. (2013) Acupuncture and herbal medicine used for treatment of anhidrosis. J. Vet. Intern. Med. 27, 656-656.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Mallicote, M.F., Medina, C.I., Xie, H., Zilberschtein, J., Atria, S., Manzie, M., Hernandez, J. and MacKay, R.J. (2013) Acupuncture and herbal medicine used for treatment of anhidrosis. Submitted to Equine Veterinary Journal