Trouble for Horses in Paradise: Toxicity and Fatality Resulting from the Consumption of Indigofera spicata (Fabaceae) on Oahu Island

This is the first account of fatal toxicity in horses resulting from grazing on the pasture plant creeping indigo, Indigofera spicata, on the island of Oahu, in the Hawaiian Islands. A survey in the town of Waialua on the north shore of Oahu island indicated that creeping indigo is common and abundant on grazing lands during the rainy season and requires intensive chemical and physical control measures. Four pastures were surveyed where ranchers reported mortality of more than 17 horses since 2020. We document these incidents to alert state animal and livestock support officials, groups monitoring invasive species, and horse owners regarding the problem of this noxious weed and to support breeders with information to confront its invasiveness. Herbicide treatment is not economically feasible, and breeders opted to physically uproot the plants from the paddocks and restrain horses to clear pastures as they were eliminating the plants. We urge state officials for a long-term control strategy to reduce the problems associated with this weed.


Background
Creeping indigo, Indigofera spicata Forsskal (Fabaceae) is a perennial herb native to tropical Africa, Madagascar, and the Mascarene islands, with widespread prevalence in grazing pastures throughout tropical Asia and Australia [1]. In Hawaii, it was introduced prior to 1929 as a pasture legume, and soon appeared to be poisonous to livestock. Creeping indigo is an aggressive, drought-tolerant, relatively palatable prostrate legume. Currently, it is adapted to low elevation, dry, and disturbed areas on all major Hawaiian Islands [2,3].
Creeping indigo has become invasive in many regions where it was introduced as a forage or cover crop, such as in Puerto Rico and southern Florida [4]. Because it is highly appealing to livestock, horses may be poisoned after eating large amounts of the legume. It carries a high level of toxic alkaloids that can cause miscarriage in pregnant cattle, among other effects [5,6].

Plant Identity, Origin, and Distribution
Plants of the tribe Indigofereae (Fabaceae) are often very attractive, pink-to red-flowered shrubs and herbs. The largest genus is Indigofera (≈700 species) which is pantropical, with two-thirds of the species restricted to Africa, the rest in the Sino-Himalayan subregion, Indonesia, and Brazil. Because of the complex chemistry of this genus, it contains many species that are poisonous or important in agroindustry [2]. For instance, the original source of blue indigo dye is Indigofera tinctoria L., a shrub cultivated in tropical and temperate Asia and Africa [1]. many species that are poisonous or important in agroindustry [2]. For instance, the original source of blue indigo dye is Indigofera tinctoria L., a shrub cultivated in tropical and temperate Asia and Africa [1].
I. spicata, also known as lawn indigo or trailing indigo, is an herbaceous plant with prostrating, slightly flattened and sparsely hairy stems that branch out from a central root. The leaves are usually light to dark green in color, with 5-11 oblong leaflets per stem arranged in an alternating pattern (0.5-3.0 cm long) with sparsely hairy upper surfaces and dense hairs on the undersides. The flowers are small, red or pink, and pea-shaped (0.5 cm long), arranged in elongated clusters up to 10 cm long, and produce abundant narrow, cylindrical, straight, sharp-tipped seed pods (1.5-3.5 cm long) (Figures 1 and 2). This species reproduces mainly by seeds dispersed by lawn mowers, contaminated soil, water, and mud attached to animals and vehicles. It is a very common weed of lawns, gardens, roadsides, disturbed sites, and waste areas [2].  central roots that are difficult to kill with conventional herbicides and must be dug out. Creeping indigo also spreads by its long, hard-to-pull roots and seeds, making it difficult to fully kill it in one round.
I. spicata was valued as a cover crop to manage soil quality in coffee lands in Africa and was introduced into India, Java, Malaya, and the Philippines as an ornamental ground cover crop in tea, rubber, oil palm, and sisal plantations [1]. It has also been introduced to Australia, several Pacific islands, central America, and the Caribbean islands [1-3]. I. spicata was valued as a cover crop to manage soil quality in coffee lands in Africa and was introduced into India, Java, Malaya, and the Philippines as an ornamental ground cover crop in tea, rubber, oil palm, and sisal plantations [1]. It has also been introduced to Australia, several Pacific islands, central America, and the Caribbean islands [1-3].

Symptoms, Toxicity, and Toxic Components
Creeping indigo grows very close to the ground, which makes it difficult to find it within a pasture ( Figure 1A). Beside growing from its seeds, it grows back from its long taproots, which makes it hard to destroy it with a single application of chemicals ( Figure  1B) [1,2].
Two putative toxins found in creeping indigo are 3-nitropropionic Acid (3-NPA) and indospicine. The highly toxic 3-NPA is a potent and irreversible inhibitor of mitochondrial succinate dehydrogenase, a key enzyme in transforming glucose and oxygen into useable energy, accounting for the early and prominent neurologic signs of toxicity. Because 3-NPA is metabolized quickly, it is unlikely to be found when testing the serum of affected animals. It is associated with motor disorders in livestock and humans when ingested [7,8].
Indospicine is a non-protein amino acid toxic to the liver because of its antagonism to arginine, an essential α-amino acid that is important in the biosynthesis of proteins. One of its principal toxic actions is the inhibition of nitric oxide synthase, an action associated with the development of ulceration of mucous membranes [9], Figure 3B. Although horses are relatively resistant to the liver-damaging effects of indospicine, this toxin accumulates in the tissues of horses dying from the disease. Indospicine accounts for ≤0.5% of the dry matter of creeping indigo and can be detected in the serum of affected animals [10]. It is highly toxic to livestock in small doses, causing loss of vitality, liver degeneration, and abortion in cattle and goats. It is particularly dangerous to horses, which relish plants containing it [10,11]. Horses need to eat around 4.5 kg of creeping indigo daily for

Symptoms, Toxicity, and Toxic Components
Creeping indigo grows very close to the ground, which makes it difficult to find it within a pasture ( Figure 1A). Beside growing from its seeds, it grows back from its long taproots, which makes it hard to destroy it with a single application of chemicals ( Figure 1B Two putative toxins found in creeping indigo are 3-nitropropionic Acid (3-NPA) and indospicine. The highly toxic 3-NPA is a potent and irreversible inhibitor of mitochondrial succinate dehydrogenase, a key enzyme in transforming glucose and oxygen into useable energy, accounting for the early and prominent neurologic signs of toxicity. Because 3-NPA is metabolized quickly, it is unlikely to be found when testing the serum of affected animals. It is associated with motor disorders in livestock and humans when ingested [7,8].
Indospicine is a non-protein amino acid toxic to the liver because of its antagonism to arginine, an essential α-amino acid that is important in the biosynthesis of proteins. One of its principal toxic actions is the inhibition of nitric oxide synthase, an action associated with the development of ulceration of mucous membranes [9], Figure 3B. Although horses are relatively resistant to the liver-damaging effects of indospicine, this toxin accumulates in the tissues of horses dying from the disease. Indospicine accounts for ≤0.5% of the dry matter of creeping indigo and can be detected in the serum of affected animals [10]. It is highly toxic to livestock in small doses, causing loss of vitality, liver degeneration, and abortion in cattle and goats. It is particularly dangerous to horses, which relish plants containing it [10,11]. Horses need to eat around 4.5 kg of creeping indigo daily for about two weeks to develop signs of toxicity. Symptoms vary but can include runny squinting eyes, sleepiness, involuntary rapid and repetitive movements of the eyes, abnormal gait, and mild colic-type signs. Special tests on postmortem tissues can identify the toxin [12][13][14]. about two weeks to develop signs of toxicity. Symptoms vary but can include runny squinting eyes, sleepiness, involuntary rapid and repetitive movements of the eyes, abnormal gait, and mild colic-type signs. Special tests on postmortem tissues can identify the toxin [12][13][14]. Ossedryver et al., 2013 [15], reported that horses that continuously grazed a pasture containing about 24% I. spicata for 4-6 weeks developed weakening, lack of muscle coordination, depression, swallowing disorder, excessive flow of saliva, and bad breath. One horse in the study recovered with supportive treatment, but two other horses were hopelessly sick. Their livers showed lymphocytic infiltrations and hydropic degeneration of hepatocytes, with mild to moderate liver necrosis. Indospicine was detected in the serum, heart, and liver of euthanized horses. 3-NPA and indospicine toxins were believed to have caused neurological syndrome and the formation of indospicine residues in the tissues of animals grazing paddocks infested with I. spicata in Australia [15].

Signs of Creeping Indigo Toxicity
Early neurological signs include a change in performance in affected horses, with individuals becoming less energetic than usual and showing degrees of sleepiness ranging from mild exhaustion to loss of consciousness as the condition progresses over days to weeks. Horses also show episodes of standing sleep-like activity, head-pressing into corners, or compulsive walking around the inside of a stall or paddock. Some affected horses have been seen with their heads tilted to one side and their necks and bodies twisted in the same direction, indicating involvement of the balance centers of the brain. Most horses Ossedryver et al., 2013 [15], reported that horses that continuously grazed a pasture containing about 24% I. spicata for 4-6 weeks developed weakening, lack of muscle coordination, depression, swallowing disorder, excessive flow of saliva, and bad breath. One horse in the study recovered with supportive treatment, but two other horses were hopelessly sick. Their livers showed lymphocytic infiltrations and hydropic degeneration of hepatocytes, with mild to moderate liver necrosis. Indospicine was detected in the serum, heart, and liver of euthanized horses. 3-NPA and indospicine toxins were believed to have caused neurological syndrome and the formation of indospicine residues in the tissues of animals grazing paddocks infested with I. spicata in Australia [15].

Signs of Creeping Indigo Toxicity
Early neurological signs include a change in performance in affected horses, with individuals becoming less energetic than usual and showing degrees of sleepiness ranging from mild exhaustion to loss of consciousness as the condition progresses over days to weeks. Horses also show episodes of standing sleep-like activity, head-pressing into corners, or compulsive walking around the inside of a stall or paddock. Some affected horses have been seen with their heads tilted to one side and their necks and bodies twisted in the same direction, indicating involvement of the balance centers of the brain. Most horses that continue to consume the plant eventually become unconscious or develop convulsions and mild liver pathology [13,14].
Non-neurologic signs may include weight loss, high heart and respiratory rates, labored breathing, high body temperature, foaming from the mouth, dehydration, pale mucous membranes, watery discharge from the eyes and squinting, light sensitivity, corneal ulceration and severe ulceration of the tongue and gums, and acute diarrhea [16], Table 1, Figure 3A,B.   10 27.45 W, elevation 6.0 m), reported horse deaths and illnesses on the North Shore, likely from creeping indigo feeding. He also testified regarding three horses currently experiencing toxicity and elevated liver function tests, and the loss of one horse from liver failure in 2017 (Taylor Mower, Dillingham Ranch Manager, personal communication). Four pastures were surveyed where ranchers reported added mortality of more than 17 horses since 2020.

Chemical Control
Although there is no commercial indigo-selective herbicide recommended for creeping indigo to kill leaves and roots, two herbicides containing aminopyralid are used, with necessary retreatment the following year. One of these is Milestone (DOW AgroSciences) at 5 fluid ounces per acre. Hawaii Polo Club on Oahu uses GrazonNext HL DOW herbicides for control of broad-leaf weeds on rangelands at 24 fluid ounces (= 0.7 L) per acre [17]. Plants die if sprayed with Grazon or other similar products, but pulling up the dead plants is necessary since the seeds are still viable (Figure 2). The dead plants must be removed and disposed [18].

Treatment and Prevention
There is no effective treatment for advanced toxicity by creeping indigo, only the management of symptoms is possible. Horses that are quickly removed from infested sites often recover completely, but gait abnormalities sometimes persist. Because of their relative resistance to indospicine, horses lack substantial liver lesions. Combined with the neurological disease that results from 3-NPA poisoning, this suggests that L-arginine alone would have little benefit in the treatment of the neurological symptoms [19]. Thiamine was also cited in the literature as a current treatment for nitro toxicity in livestock, but research showed this treatment is ineffective [19]. Therefore, the management of affected horses should emphasize their removal from the source of toxicity to prevent any injuries. It was suggested that livestock poisoning by I. spicata can be prevented by reducing the weed population to <20% of the total forage available, but recent evidence does not support this view. The confinement of horses to clean paddocks where creeping indigo is removed by physical means or herbicide application is the best control measure [13,20].

Discussion
Several species of the genus Indigofera (e.g., I. hirsute L., I. pilosa Poir., I. schimperi Jaub. & Spach, and I. subulate Vahl ex Poir.) have shown great potential as high-protein grazing forage, green manure, or cover crops for livestock in Africa and Asia [21,22]. Some Indigofera species are known to contain the pigment indigo used for commercial dye production. However, reservations regarding the toxicity of I. spicata have restricted its establishment as a feed supplement [21]. It was introduced to Hawaii as a pasture legume to supplement the diets of grazing animals, but its toxicity when consumed in large quantities was unknown before importation.
The introduction of the plant and seeds to Hawaii at that time was lawful, with no detailed risk analysis by the Animal and Plant Health Inspection Service (APHIS). Currently, creeping indigo is thriving in low-elevation, dry, disturbed areas [2]. It was Vet. Sci. 2022, 9, 271 7 of 9 initially introduced under the name I. hendecaphylla Jacq. [23], a common species, distributed throughout the Old-World tropics and subtropics to the Pacific Islands, whereas I. spicata is confined to Africa, Yemen, Madagascar, and the Mascarenes [1]. Previous reports on the toxicity of creeping indigo as a cattle fodder referred to I. hendecaphylla [23,24]. I. spicata has also been shown to be hepato-toxic when grazed by cattle [5]. The free, non-protein amino acid indospicine was detected in the seed and leaf material of this plant. Besides I. spicata, toxicity was also reported for the African species I. hirsute L. and I. linifolia (L.f.) Retz. [21,22].
The enzyme gamma glutamyl-transferase (GGT) is generally found in liver cell membranes and biliary epithelial cells. Increased blood levels of GGT indicate that something is damaging the liver, though they do not identify the specific problem ( Table 2). Increased levels of GGT can take several weeks before returning to normal. Higher-than-normal test results could be a sign of liver disease such as hepatitis, cirrhosis, tumors, or pancreatic cancer [25,26].
The Hawaii Polo Club had multiple cases of suspected alkaloid toxicity in horses. Field inspection of four pastures revealed that the original suspect was I. spicata mixed with the nutritious kikuyu grass, Cenchrus clandestinus Hochst. ex Chiov. (Poaceae). Other invasive weeds were common in paddocks but disregarded as the culprit of horse toxicity

Conclusions
This synopsis of the invasive plant I. spicata on Oahu island indicates a recent increase in toxicity and fatality in horses on four ranches. The plant is widespread on pasture lands of windward Oahu during the rainy season. Chemical control with broad-spectrum herbicides appears ineffective, and manually pulling the plants is not sufficient for managing this weed. In general, a single herbicide application will not suppress creeping indigo permanently; research on chemical control is essential for new selective herbicides that kill leaves and taproots and do not injure useful pastures.
We urge state officials and Regulatory Enforcement and Animal Care (REAC) program USDA-APHIS to fund research on long-term solutions to decrease the plant population. Creeping indigo is very palatable to horses, and it is important for horse owners to recognize the plant and know the signs of toxicity and ways to prevent it from being consumed. Statewide surveys and outreach programs are needed to accurately determine the infestation levels of I. spicata and unreported livestock toxicity. This weed should be elevated to the top list of noxious weeds targeted for management in Hawaii. The Hawaii Department of Agriculture may participate in a subsidy program to assist ranchers in chemical control to mitigate this problem until biological control agents are found. A biological control endeavor in the native region is recommended for importation of host-specific natural enemies to reduce its invasiveness. For instance, the wild indigo weevils, Trichapion rostrum (Say), (Coleoptera: Brentidae), and Tychius sordidus LeConte, (Coleoptera: Curculionidae), are seed predators used in biological control and can adversely affect the reproduction of related herbaceous Fabaceae plants [28]. Several other natural enemies (i.e., insects, nematodes, and diseases) of Indigofera can be examined for specificity.