Source: UNIVERSITY OF FLORIDA submitted to NRP
CREEPING INDIGO: AN EMERGING THREAT TO ANIMALS IN FLORIDA AND OTHER SOUTHEASTERN STATES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1011516
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Feb 6, 2017
Project End Date
Feb 5, 2021
Grant Year
(N/A)
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
Creeping indigo poisoning is an emerging problem among horses in Florida and border southeastern states. The plant likely entered Florida before 1940 as a result of separate accidental introductions in Gainesville and Dade county. It has subsequently naturalized and spread throughout Florida and into neighboring states. Individual cases and small outbreaks of CI poisoning in horses, donkeys, and ponies have been recognized from about 1980 in Dade Country and since 2000 along the I-4 corridor with occasional cases in north-central Florida and the panhandle. Because it is a ground-hugging plant that expands outward by runner stems from a low central crown, CI is difficult to spot under the leaves of pasture grasses. Horses apparently enjoy the taste of CI and may even seek it out in preference to grass. Poisoning causes serious signs of nervous system collapse with signs such as blindness, depression, aimless wandering, stumbling, falling down and convulsions. In addition to the nervous signs, affected horses rapidly lose weight and there are often deep wide ulcers on the tongue, gums, lips and eyes. The eyes also may be cloudy and squint and tear when the poisoned horse is in direct sunlight. There is no antidote for CI poisoning and even with excellent care affected horses often die or are permanently affected. At least 300 horses had died in Dade County alone by 1988, when the cause was first suspected and numerous cases have been reported in recent years in central and north Florida.There are two potent toxins in CI that cause the signs described above: IND and NPA. IND causes the non-nervous effects of CI, including weight loss and ulcers. IND typically is 0.05-0.15% of the dry weight of CI plants in Australia and causes liver and eye damage when given to experimental animals. Interestingly, horses appear to be resistant to liver damage. IND can be measured in the blood of animals grazing CI and over time may build up to very high concentrations in muscle and liver. NPA, rather like cyanide, kills energy-producing mechanisms in the brain. Brain cells have no energy stores so are quickly damaged and die when poisoned by NPA, leading to the nervous system collapse described above. The toxin accounts for up to 0.27% of dry weight of CI. NPA is found in blood immediately after it is given into the stomach, but it is eliminated from the blood within a couple of hours.We want to use state-of-the-art tests for IND and NPA to determine the amount of these toxins in CI plants and in the blood of horses eating them and in this way we hope to determine the extent of the problem of CI poisoning in Florida. We will proceed as follows: first, we will establish the assays for IND and NPA on the high-tech analytical equipment used by the Center for Human and Environmental Toxicology at the University of Florida; second, we will use these assays to measure IND and NPA in CI plants from various Florida locations and seasons; third, we will measure IND and NPA in blood and spinal fluid from horses eating CI, and; fourth, we will measure these toxins in plants and horses during occurring outbreaks of CI poisoning around the state.We expect, after completing this work, to have described the toxic potential of CI for horses in Florida. We hope publication of these findings will both alert horse owners and veterinarians to the problem and provide the underpinnings of further investigations aimed at early detection and effective treatment of clinical cases.
Animal Health Component
95%
Research Effort Categories
Basic
(N/A)
Applied
95%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31438101150100%
Goals / Objectives
Objective 1. To adapt published assay protocols to the UPLC-MS/MS methodology used by the Center for Human and Environmental Toxicology at the University of FloridaObjective 2. To quantify indospicine and 3-nitropropionic acid in Indigofera spicata plants from various Florida locations and seasons.Objective 3. To quantify indospicine and 3-nitropropionic acid in plasma and cerebrospinal fluid from horses after a single meal of CI.Objective 4. To quantify indospicine and 3-nitropropionic acid in plants and horses associated with Indigofera spicata toxicoses.
Project Methods
1.0 To adapt published assay protocols to the UPLC-MS/MS methodology used by the Center for Human and Environmental Toxicology at the University of Florida 1.1 Methods 1.1.1 Installation and validation of assays for l-indomethacin and 3-nitropropionic acid 1.1.2 Assay of toxins in I. spicata I. spicata plants will be air dried at ambient temperature and then milled to pass a 1-mm screen. Dry matter (DM) percentage of specimens will be determined by heating to constant weight at 105°C under nitrogen. 1.1.3 Stability of toxins in I. spicata Additional extractions and toxin quantifications will be performed on plants at the following stages: immediately after collection (I.e., before drying), after rack drying, and after storage of dried specimens for 4 weeks, in order to determine the stability of the toxins during drying and storage.2.0 To use these assays to quantify indospicine and NPA in Indigofera spicata plants from various Florida locations and seasons 2.1 Methods 2.1.1 Plant collection from geographic zones in Florida.The state will be divided as shown in into 13 geographical zones on the basis of minimal annual temperature, yearly rainfall, and Köppen-Geiger climate classification. Plants will be collected from at least three locations within each zone. Six-inch plant sections will be dried for 14 days then analyzed for IND and NPA content. 2.1.2 Serial toxin analyses of individual I. spicata plants.We will identify 10 I. spicata plants around the college. Each plant will be protected. Six-inch pieces of stem will be removed every 2 mo for 1 y. Five sets of dried plants will be analyzed for toxins. 2.1.3 Quantification of indospicine and 3-nitroprionoic acid in stored plant specimensPlants will be assayed for toxins by UPLC-MS/MS.3.0 To quantify IND and NPA in plasma and cerebrospinal fluid from horses eating creeping indigo 3.1 Experimental Design Five ponies will be fed chopped dried CI such that the CI supplies IND at 1 mg/kg bodyweight daily for five days. The NPA dosage will reflect the relative concentration of the toxins in the challenge feed but likely will be 0.5 to 2 mg/kg/day. Ponies will be monitored frequently throughout the experiment and blood samples will be collected at intervals for CBC, plasma chemistries, and toxin concentrations. Ponies will be offered for sale at the completion of the experiment. 3.2 Methods 3.2.1 Plant collection for feeding experiments. The feeding experiment will require 25 - 75 kg of fresh CI. When CI was planted as a forage crop it was reported to yield 12,738 kg/hectare (1.3 kg/m), so requirements would be met by 19 to 58 m of densely growing I. spicata. We are aware of suitable stands of I. spicata in Dade county and southern Marion county that should readily supply the required amount of plant. After harvest, the CI will be dried on hanging racks for at least 14 days (as above), then chopped (~1-cm lengths) and stored in sealed containers at -20 C until used. Representative samples of CI will be assayed before storage, then each aliquot used for a feeding experiment will be retested immediately before the experiment to allow precise adjustment to the required IND dose. 3.2.2 Animals. Experimental subjects will be adult ponies purchased for the study (~250 kg/pony). Ponies will be enrolled only if they have body condition scores of at least 4/9 and are healthy on the basis of normal results of a physical examination and CBCs and biochemical profiles performed less than 2 weeks before experiments. 3.2.3 Creeping indigo/alfalfa meals. The daily IND (and NPA) dose will be fed in 2 meals, given 12 hours apart. Alfalfa hay will be chopped at 1-2 cm and mixed with chopped dried CI to total weight of 3 g/kg (0.2%) BW per feeding and offered to subject ponies in a feed tub morning and evening. With projected IND concentrations of 0.5 to 1.5 mg/g DW, CI will daily comprise from 0.7 to 2 g/kg of this mix (0.35 - 1 g/kg/meal). 3.2.4 Experimental Procedure Each pony will be weighed, brought into a stall in the afternoon, and given free access to water and a small amount of hay. The next morning, an indwelling 5.25-in catheter will be secured in the left jugular vein. This catheter will be regularly flushed with sterile saline and used for all blood withdrawals. The designated CI/alfalfa mix will be left in front of the pony for 6 h after each feeding. Any feed remaining will be homogenized in a blender with water and given by stomach tube. Twenty-four hours after the last feeding, ponies will be turned out in paddocks free of CI for an additional 28 days of monitoring. The following procedures will be performed during the observation period:Clinical SignsWeighing and Pasture Assessment c. Blood Samples - CBC (2-mL EDTA vacutainer tube) and Chemistry Panel (4-mL Li heparin tube) - before feeding (time = 0 h), every 2 d while in stalls, then once weekly after turnout; Indospicine analyses - (10-mL Li heparin tube) - before first CI feeding, then daily (before AM feeding) while in stalls and weekly thereafter. 3-nitropropionic acid analyses - (10-mL Li heparin tube) - before CI feeding (time = 0), then 2, 4, 6, and 8 h after beginning the morning CI feeding on days 1 and 5. Blood tubes for toxin analyses will be stored in ice (for maximum of 4 h), then plasma separated by centrifugation and stored in 1-mL aliquots at -80 C. d. Feeding - After each CI/alfalfa meal, ponies will be given a weighed amount of coastal Bermuda hay (approximately 4.5 g/kg BW). Once on pasture, ponies will be fed coastal Bermuda hay (1.5% BW, divided in two daily feedings). e. Collection of Cerebrospinal fluid4.0 To quantify IND and NPA in plants and horses associated with naturally occurring Indigofera spicata toxicoses. 4.1 Experimental Design Naturally occurring cases of CI poisoning in Florida will be investigated by our research group during the year of study. Naturally occurring cases of CI poisoning will meet the following criteria for case definition: (1) Neurologic abnormalities and the presence of at least one non-neurologic sign (photophobia, corneal opacity, corneal, oral, lingual ulcers, or weight loss) (2) Presence of identified creeping indigo on the pasture where the affected animal is housed. 4.2 Methods Outbreaks and individual cases of suspected CI toxicity in equids will be logged by our research group via the following methods (1) self-reporting on an interactive Florida map on our planned Creeping Indigo Toxicity Resource webpage; (2) email blasts to our mailing lists of equine veterinarians and clients in Florida and south Georgia, and; (3) information and referral calls from horse owners and veterinarians. Each premise with an affected horse or horses will be visited by a team including at least one of the applicants and the following data collected: Grazing, feeding, and clinical histories of affected and unaffected animals; description of pasture areas and pasture species for entire premise; clinical signs of affected animal(s) including full neurologic examination and video documentation; presence of I. spicata in forage available to affected animals (yes/no); percentage (by weight) of CI in foliage within a rectangular frame repeated along a transect of the pasture. Blood samples will be collected from the following cohorts of animals: (1) equids with signs of CI toxicosis (maximum of 5 per premise); (2) all clinically normal equids of all ages grazing the implicated pasture(s) (maximum of 3 per neurologic case); (3) representative non-equid livestock grazing the implicated pasture (maximum of 5 per premise); (4) clinically normal horses from adjacent pastures lacking CI (on same or neighboring premises) (maximum of 3 per neurologic case). Bloods will be processed for toxin analyses and submitted for CBCs and biochemical profiles. All plasmas and representative dried CI plants will be assayed for IND and NPA.

Progress 02/06/17 to 02/05/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training in experimental design and research method for two large animal medicine residents How have the results been disseminated to communities of interest?For equine veterinarians: publication in most widely distributed equine veteinary journal (Equine Veterinary Journal) What do you plan to do during the next reporting period to accomplish the goals?This is final report

Impacts
What was accomplished under these goals? Objective 1. Completed, reported in publication cited above Objective 2. Partially completed. 100 kg of creeping indigo plants from around north central florida have been harvested and representative samples have been assayed for toxins. Objective 3. Completed and reported in publication cited above Objective 4. Completed and reported in publication cited above and in another publication in preparation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Darby S, Sanchez LC, Mallicote MF, House AM, Plummer CE, Nadruz V, Benmoha RH, Roberts SM, Derendorf H, Silva-Sanchez C, Claire J, MacKay RJ. Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo: Comparison with results from an episode of presumptive creeping indigo toxicosis. Equine Vet J. 2020 Dec 28. doi: 10.1111/evj.13415. Epub ahead of print. PMID: 33369767.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Equine veterinarians and horse owners in the state of Florida Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Yes, they are being submitted for publication in the Equine Veterinary Journal, the most widely disseminated journal for equine veterinarians and widely read by those in Florida. The results were submitted preliminary to veterinary internists by presentation at the ACVIM conference. Also, results have been published within our updating creeping indigo review on the UF College of Veterinary Medicine website. What do you plan to do during the next reporting period to accomplish the goals?Will supervise the submitted publication through the rest of the review and publication protest. My team will investigate further instances of creeping indigo intoxication and continue to update the website. I will be retiring in May 2021.

Impacts
What was accomplished under these goals? Objective 1 and Objective 3 were completed and partially reported in the 2017 report and the results have been submitted for publication and cited herein ( Plasma l-indospicine and 3-nitropropionic acid inponies fed creeping indigo: Comparison with results from an episode of presumptive creeping indigo toxicosis). Objective 2. Approximately 105 kg of the aerial portions of creeping indigo plants were harvested from 38 locations in Alachua, Levy, and Marion counties. These results are included in the publication that is in review and cited in this report. Objective 4. An investigation was undertaken of an episode of fatal creeping indigo toxicity in Marion County that involved 6 horses, 3 of whom were humanely euthanized in extremis. The results are reported in the above publication.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Darby S, Sanchez LC, Mallicote MF, House AM, Plummer CE, Nadruz V, Benhoma RH, Roberts SM, Derendorf H, Silva-Sanchez C, Claire J, MacKay RJ. Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo: Comparison with results from an episode of presumptive creeping indigo toxicosis. Equine Vet J, In Review
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Darby, S.M., Mallicote, M., Sanchez, L.C., House, A., Plummer, C., Benmoha, R., MacKay, R. Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo. Abstract E06. J. Vet. Intern. Med. 33, 2425:33, 2019


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Veterinary toxicologists, equine veterinarians, and horse owners Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Veterinary students have learned to perform complete physical examinations and blood collections in ponies. A staff member in the toxological laboratory has developed, validated, and optimized two new analytical assays for the laboratory. How have the results been disseminated to communities of interest?The results of the work so far performed under objectives 3 and 4 have been presented to local veterinarians at the Ocala Equine Conference and RDVM Day here at the University of Florida, and to veterinary internists at the American College of Veterinary Medicine's annual forum. A manuscript is in preparation that will be send to the Equine Veterinary Journal. What do you plan to do during the next reporting period to accomplish the goals?Submit the manuscript mentioned in the previous section. Continue investigating natural cases of creeping indigo toxicity.

Impacts
What was accomplished under these goals? Objective 1 and Objective 3 were completed and reported in previous progress report. Objective 2. Because of funding shortfall, no progress was made under this objective. Objective 4. A farm investigation was made on a small outbreak of creeping indigo toxicosis in Marion County. This investigation included quantification of indospicine and 3-nitropropionic acid in blood from 3 horses that died and 4 horses that did not develop clinical signs.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Darby SM, Mallicote M, Sanchez LC, House A, Benmoha R, MacKay R. Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo. J Vet Intern Med. p32, Abstract E06, https://doi.org/10.1111/jvim.15597


Progress 02/06/17 to 09/30/17

Outputs
Target Audience:Veterinary toxicologists, equine veterinarians, and horse owners Changes/Problems:Budget cutback prevented performing experiments described under Objective 2 and delayed the start of experiments described under Objective 4. Completed experiments were performed as described in the IACUC application attached to the original proposal. No major changes were made to that experimental design. What opportunities for training and professional development has the project provided?Veterinary students have learned to perform complete physical examinations and blood collections in ponies. A staff member in the toxological laboratory has developed, validated, and optimized two new analytical assays for the laboratory. How have the results been disseminated to communities of interest?The study has not been completed yet. The assays under objective 3 will be completed during the spring and the results will be presented for the first time at the UF College of Veterinary Medicine's annual RDVM (referring veterinarian's) conference to be held June 8. The results likely will be published in the journal Toxicon. What do you plan to do during the next reporting period to accomplish the goals?Complete the assays needed for Objective 3. Begin investigating natural cases of creeping indigo toxicity to meet the objectives in Objective 4.

Impacts
What was accomplished under these goals? Objective 1: Assays for l-indospicine and 3-nitropropionic acid were established successfully and used for assay of these toxins in creeping indigo plants and plasma and cerebrospinal fluid samples from horses. Objective 2: No progress to report under this objective Objective 3: Plant Collection. A total of 99 kg of creeping indigo (Indigofera spicata) plants were collected from 37 locations within approximately 50 miles of the University of Florida. Milled samples of plants were dried for 10 days on drying racks to 26 kg (30% of wet weight), chopped and stored at -20 C. Milled samples of 5 of the collections were assayed for l-indoscpicine (Indo) and 3-nitropropionic acid (3-NPA) by ultra performance liquid chromatography-tandem mass spectroscopy and yielded concentrations of 1520 to 3500 ug/g for Indo and 6 to 30 ug/g dry matter for 3-NPA. Two of these lots were blended to provide final concentrations of 1900 and 10 ug/g INDO and 3-NPA, respectively. Animal Feeding Experiment. Five healthy ponies were fed daily (in 2 feedings) 1 mg/kg INDO and 5 ug/kg 3-NPA as a mixture of chopped CI and chopped alfalfa (6 g/kg daily). Additional coastal bermuda hay was fed to provide total of 1.5% of body weight of forage daily. Ponies were fed this mixture in stalls for 5 days. 24 h after completing CI feeding, ponies were turned out to pasture for a further 28 days. Clinical signs were monitored throughout and blood was collected as follows:CBC (2­mL EDTA vacutainer tube) and Chemistry Panel (4­mL Li heparin tube) - before feeding (time = 0 h), every 2 d while in stalls, then once weekly after turnout? Indospicine analyses ­ (10 ­mL Li heparin tube) - before first CI feeding, then daily (before AM feeding) while in stalls and weekly thereafter. 3-­nitropropionic acid analyses ­ (10­mL Li heparin tube) - before CI feeding (time = 0), then 2, 4, 6, and 8 h after beginning the morning CI feeding on days 1 and 5.24 h after the last CI meal, 10 mL of cerebrospinal fluid (CSF) was collected by aspiration from the lumbosacral site of each pony. Results. Clinical Signs. Ponies remained healthy throughout the study and no significant changes were noted in CBC and plasma chemistries. Plasma indospicine. Indo concentrations rose steadily from day 1 (before CI feeding, 36 ± 29 ng/mL) to day 6 (24 h after CI feeding, 3533 ± 616 ng/mL), then declined over the subsequent 28 days after CI feeding was discontinued to 1413 ± 318 ng/mL. Plasma 3-NPA. 3-NPA analyses have only just begun. Only the time 0 (before feeding) and 2-h post-feeding samples have been analyzed from day 1 (the first day of feeding). On the basis of published information for livestock, we predicted that the 2 h timepoint would contain maximal toxin concentration. Concentrations of NPA increased from 8 ± 1.5 ng/mL to 50 ± 17 ng/mL. These analyses will be completed over the next several months as funds become available.

Publications