Abstract
We investigated the health of Tokara horses (n=34) on Nakanoshima as part of conservation efforts for this breed. Although the horses were clinically healthy, elevated white blood cell count, neutrophil count and gamma-glutamyl transferase (GGT) levels were observed, leading to suspicion of chronic hepatic dysfunction and infectious disease. A survey revealed that the caretakers had been feeding the horses Leopard plant, which contains toxic pyrrolizidine alkaloids (PAs). The abnormal GGT levels observed in these horses may suggest the possibility of chronic PA poisoning resulting from the consumption of this plant. These findings suggest that raising awareness of poisonous plants and carefully managing plant species in grazing areas could enhance the conservation of native horse breeds.
Keywords: hepatotoxicity, Leopard plant, pyrrolizidine alkaloid, Tokara horse
The Tokara horse is one of eight native equine breeds in Japan (the others are Hokkaido, Kiso, Misaki, Noma, Taishu, Miyako, and Yonaguni horses). These breeds are registered as native to Japan by the Japan Equine Affairs Association (JEAA). Currently, Tokara horses are raised at three locations: Nakanoshima, an island in the Tokara chain, and Kaimon Foothills Nature Park and the Kagoshima University Experimental Farm in Iriki, on the Kyushu mainland, but only the Tokara horses on Nakanoshima have been registered as a natural treasure by Kagoshima Prefecture.
Conservation remains a critical issue for the Tokara horse. According to JEAA figures, the population of this breed declined from 130 horses in 2017 to 79 in 2021, although it has since rebounded slightly, reaching a total of 90 in 2023. Furthermore, the Tokara horse also faces the problem of declining genetic diversity [23]. To address these conservation issues, we previously investigated the health of Tokara horses in Kaimon Foothills Nature Park (hereafter, Kaimon horses), focusing mainly on hematological and blood biochemical data [26]. Similar investigations of the Nakanoshima and Iriki populations are also essential for the overall conservation of the Tokara horses.
The aim of this study was to assess the health status of the Tokara horses on Nakanoshima (hereafter, Nakanoshima horses) through hematological and blood biochemical investigations, and to identify conservation challenges and potential health risks for these horses through interviews with their caretakers and a survey of their pasture and the surrounding area.
We targeted Nakanoshima horses undergoing physical examination evaluation and blood sampling for hematology and biochemistry in 2022, 2023, and 2025, for evaluation in this study. We excluded horses under two years of age and those for which individual information was unavailable. A total of 34 horses were enrolled in the study. This study population consisted of 12 horses examined in 2022, nine horses in 2023, and 13 horses in 2025. By sex, the population included 13 males and 21 females, broken down by year as follows: five males and seven females in 2022, three males and six females in 2023, and five males and eight females in 2025. The study population had an age range of 3–17 years (mean ± standard deviation [SD]: 6.9 ± 3.5 years), broken down as follows: 6.8 ± 3.5 years in 2022, 7.3 ± 3.4 years in 2023, and 6.7 ± 3.9 years in 2025. Each horse was physically examined at the time of blood sampling. All blood samples were collected by jugular venipuncture, in October 2022, October 2023, or August 2025, and transferred into tubes containing dipotassium ethylenediaminetetraacetic acid (EDTA-2K; VP-DK052K, TERUMO, Tokyo, Japan) and plain tubes (VP-P100K, TERUMO). Serum was separated by centrifugation (3,000 g, 15 min) and stored at −80°C until measurement.
Hematological parameters were measured using a commercially available automated cell counter (pocH™-100iV Diff, Sysmex, Kobe, Japan). Differential leukocyte counts were determined by microscopically examining Giemsa-stained blood smears. Serum biochemical analysis was performed using a commercially available automated analyzer (DRI-CHEM 7000V, FUJIFILM, Tokyo, Japan). Serum amyloid A (SAA) was quantified by the latex aggregation method (LZ test “Eiken” SAA; Eiken Chemical, Tokyo, Japan) using equine standard serum with SAA concentrations ranging from 0.0 to 400.0 µg/mL; these standards were produced by methods described in a previous report [15]. The detection limit for SAA was less than 0.1 µg/mL. Data are expressed as mean ± SD and median. We also compared these test results with previously reported data we obtained from Kaimon horses [26].
We also conducted interviews with the caretakers regarding horse care and management. We then surveyed the pasture and its surroundings to check for the presence of invasive plants.
This study utilized blood test results from the 2022 survey, which were obtained with the owners’ consent and at their request. The subsequent surveys conducted in 2023 and 2025 were approved by the Animal Care and Use Committee of Kagoshima University (KVH230009; clinical research).
All the Nakanoshima horses in this study were regarded as clinically healthy, based on physical examinations. These horses showed satisfactory vital signs and appetite, with body condition scores around 4–5. The hematological and biochemical data obtained in this study are presented in Table 1.
Table 1. Hematological and blood biochemical parameters of Tokara horses.
| Variable | Nakanoshima | Kaimon (n=90) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 2022 (n=12) | 2023 (n=9) | 2025 (n=13) | All (n=34) | |||||||
| Mean ± SD | Median | Mean ± SD | Median | Mean ± SD | Median | Mean ± SD | Median | Mean ± SD | Median | |
| White blood cells (102/µL) | 155.1 ± 22.4 | 158.0 | 147.1 ± 20.0 | 144.0 | 155.8 ± 29.1 | 158.0 | 153.2 ± 24.2 | 155.0 | 88.0 ± 15.9 | 90.0 |
| Red blood cells (104/µL) | 889.5 ± 92.7 | 895.5 | 908.7 ± 66.1 | 898.0 | 954.9 ± 131.2 | 952.0 | 919.6 ± 105.1 | 912.0 | 783.3 ± 87.0 | 777.0 |
| Hemoglobin (g/dL) | 14.5 ± 1.6 | 14.4 | 14.9 ± 1.0 | 14.8 | 14.6 ± 1.7 | 14.7 | 14.7 ± 1.5 | 14.6 | 11.8 ± 1.1 | 11.5 |
| Hematocrit (%) | 40.9 ± 4.3 | 40.1 | 42.2 ± 2.6 | 42.6 | 42.4 ± 4.5 | 42.7 | 41.8 ± 4.0 | 42.2 | 33.7 ± 3.1 | 32.5 |
| Neutrophils (102/µL) | 120.1 ± 20.9 | 111.8 | 116.8 ± 20.5 | 116.1 | 115.7 ± 30.4 | 112.7 | 117.5 ± 24.2 | 112.8 | 67.5 ± 14.9 | 69.8 |
| Lymphocytes (102/µL) | 23.3 ± 6.8 | 22.3 | 16.6 ± 3.6 | 15.8 | 28.0 ± 7.0 | 27.1 | 23.3 ± 7.6 | 22.2 | 14.5 ± 4.0 | 13.8 |
| Monocytes (102/µL) | 5.6 ± 3.4 | 5.4 | 6.6 ± 3.6 | 5.8 | 5.0 ± 2.1 | 4.4 | 5.6 ± 3.0 | 4.8 | 2.7 ± 1.3 | 2.2 |
| Eosinophils (102/µL) | 5.8 ± 3.6 | 5.2 | 6.4 ± 2.7 | 6.1 | 7.1 ± 10.2 | 3.4 | 6.5 ± 6.7 | 4.6 | 2.9 ± 2.0 | 2.6 |
| Basophils (102/µL) | 0.1 ± 0.3 | 0.0 | 0.8 ± 0.8 | 0.7 | 0.1 ± 0.2 | 0.0 | 0.3 ± 0.5 | 0.0 | 0.4 ± 0.6 | 0.4 |
| Neutrophil-to-Lymphocyte ratio | 5.6 ± 2.2 | 5.3 | 7.5 ± 2.5 | 7.2 | 4.3 ± 1.2 | 4.7 | 5.6 ± 2.3 | 5.0 | 5.0 ± 1.9 | 5.1 |
| Total protein (g/dL) | 8.6 ± 0.7 | 8.6 | 8.4 ± 0.4 | 8.3 | 8.5 ± 0.5 | 8.5 | 8.5 ± 0.6 | 8.5 | 6.3 ± 1.3 | 4.7 |
| Albumin (g/dL) | 3.1 ± 0.3 | 3.1 | 3.1 ± 0.1 | 3.1 | 2.8 ± 0.3 | 2.8 | 3.0 ± 0.3 | 3.0 | 2.5 ± 0.6 | 1.8 |
| Gamma-glutamyl transferase (U/L) | 58.7 ± 24.3 | 52.5 | 48.7 ± 14.6 | 44.0 | 90.5 ± 37.9 | 87.0 | 68.2 ± 33.2 | 56.5 | 28.5 ± 16.0 | 18.0 |
| Lactate dehydrogenase (U/L) | 662.4 ± 114.2 | 680.5 | 651.4 ± 162.8 | 584.0 | 597.7 ± 150.0 | 640.0 | 634.8 ± 140.9 | 640.0 | 470.5 ± 113.0 | 541.0 |
| Creatine phosphokinase (U/L) | 325.4 ± 182.3 | 251.5 | 471.0 ± 350.8 | 319.0 | 325.3 ± 115.5 | 323.0 | 363.9 ± 223.7 | 306.5 | 255.7 ± 108.4 | 223.0 |
| Blood urea nitrogen (mg/dL) | 18.4 ± 3.0 | 18.5 | 18.0 ± 2.0 | 18.8 | 19.5 ± 2.6 | 20.3 | 18.7 ± 2.6 | 19.1 | 17.6 ± 5.3 | 14.3 |
| Creatinine (mg/dL) | 0.9 ± 0.2 | 0.9 | 0.9 ± 0.1 | 0.9 | 0.9 ± 0.2 | 0.9 | 0.9 ± 0.1 | 0.9 | 1.2 ± 0.3 | 1.0 |
| Total bilirubin (mg/dL) | 1.5 ± 1.0 | 1.5 | 1.3 ± 0.5 | 1.5 | 1.8 ± 0.8 | 2.1 | 1.6 ± 0.8 | 1.6 | 1.0 ± 0.4 | 0.8 |
| Total cholesterol (mg/dL) | 68.1 ± 9.9 | 67.5 | 58.4 ± 9.8 | 54.0 | 57.3 ± 19.7 | 52.0 | 61.4 ± 14.9 | 60.5 | 49.8 ± 14.4 | 39.0 |
| Triglyceride (mg/dL) | 29.8 ± 26.0 | 24.5 | 20.1 ± 19.6 | 14.0 | 46.2 ± 96.3 | 18.0 | 33.5 ± 61.7 | 19.0 | 20.2 ± 22.2 | 15.0 |
| Calcium (mg/dL) | 12.1 ± 0.6 | 12.3 | 12.1 ± 0.7 | 12.2 | 13.1 ± 0.7 | 13.2 | 12.5 ± 0.8 | 12.4 | 10.3 ± 1.9 | 7.9 |
| Inorganic phosphorus (mg/dL) | 3.9 ± 0.4 | 3.9 | 3.7 ± 0.4 | 3.7 | 4.0 ± 0.6 | 4.1 | 3.9 ± 0.5 | 3.9 | 3.9 ± 0.9 | 3.9 |
| Sodium (mEq/L) | 135.6 ± 1.9 | 136.0 | 139.0 ± 1.9 | 140.0 | 138.7 ± 3.0 | 139.0 | 137.7 ± 2.8 | 137.0 | 132.7 ± 4.8 | 135.0 |
| Potassium (mEq/L) | 5.7 ± 1.2 | 5.7 | 4.8 ± 0.7 | 4.8 | 4.2 ± 0.4 | 4.3 | 4.9 ± 1.0 | 4.7 | 3.9 ± 0.8 | 4.1 |
| Chloride (mEq/L) | 99.1 ± 2.8 | 98.5 | 98.8 ± 1.8 | 99.0 | 99.0 ± 3.1 | 99.0 | 99.0 ± 2.6 | 99.0 | 93.6 ± 4.4 | 94.0 |
| Serum amyloid A (µg/mL) | 0.3 ± 0.3 | 0.3 | 5.0 ± 4.0 | 3.6 | 0.4 ± 0.6 | 0.2 | 1.6 ± 2.9 | 0.4 | 0.3 ± 2.1 | 0.0 |
The data are expressed as mean ± standard deviation (SD) and median for Tokara horses. The data for Tokara horses in Kaimon Foothills Nature Park was taken from the literature [26].
Hematology revealed elevated white blood cell (WBC) and neutrophil counts (mean ± SD [median]: 153.2 ± 24.2 [155.0] × 102/µL and 117.5 ± 24.2 [112.8] × 102/µL, respectively). These parameter values were higher than the corresponding values we previously reported for Kaimon horses (88.0 ± 15.9 [90.0] × 102/µL and 67.5 ± 14.9 [69.8] × 102/µL, respectively) [26]. Accordingly, we consider that the Nakanoshima horses may have been suffering from infectious disease. Furthermore, red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT) were also higher in the Nakanoshima horses (919.6 ± 105.1 [912.0] × 104/µL, 14.7 ± 1.5 [14.6] g/dL, 41.8 ± 4.0 [42.2]%, respectively) than in the Kaimon horses (783.3 ± 87.0 [777.0] × 104/µL, 11.8 ± 1.1 [11.5] g/dL, 33.7 ± 3.1 [32.5]%, respectively) [26].
Blood biochemistry revealed elevated gamma-glutamyl transferase (GGT; 68.2 ± 33.2 [56.5] U/L), and this value was higher than that previously reported in Kaimon horses (28.5 ± 16.0 [18.0] U/L) [26]. Accordingly, we consider that the Nakanoshima horses were experiencing hepatic dysfunction. Furthermore, total protein (TP), albumin (ALB) and total cholesterol (T-CHO) were also higher in the Nakanoshima horses (8.5 ± 0.6 [8.5] g/dL, 3.0 ± 0.3 [3.0] g/dL, 61.4 ± 14.9 [60.5] mg/dL, respectively) than in the Kaimon horses (6.3 ± 1.3 [4.7] g/dL, 2.5 ± 0.6 [1.8] g/dL, 49.8 ± 14.4 [39.0] mg/dL, respectively) [26].
Interviews with the caretakers revealed that they had been feeding the Tokara horses Leopard plant (Farfugium japonicum, Fig. 1) between April and May every year, and that the horses readily consumed this plant. The caretakers were unaware that Leopard plant was toxic to horses. In addition, it was confirmed that Leopard plant grows wild around the pasture.
Fig. 1.
Leopard plant growing wild around the Tokara horse grazing grounds in Nakanoshima.
Hematology revealed elevated levels of WBC and neutrophils in the Nakanoshima horses in this study, which surpassed the corresponding values we previously reported in Kaimon horses [26]. However, considering that the SAA level was low (median: 0.4 µg/mL), the likelihood of an active infectious disease outbreak is low. Instead, the semi-wild grazing environment of the Nakanoshima horses, physiological stress arising from handling during sampling (such as adrenaline-mediated neutrophil mobilization), or potential subclinical antigenic stimulation from parasites and chronic localized infections may have contributed to the significantly elevated WBC and neutrophil counts. This aligns with previously reported trends in many wild and free-ranging animals, which tend to exhibit higher WBC counts compared to their strictly managed, captive counterparts [1, 3, 24].
Nakanoshima horses also showed values surpassing previously reported figures for Kaimon horses in RBC, HGB and HCT levels in hematology, and TP, ALB and T-CHO levels in blood biochemistry. These findings suggest that the Nakanoshima horses were in better nutritional condition than the Kaimon horses [26]. Furthermore, the simultaneous increase in RBC, HCT, TP, and ALB levels could reflect a degree of dehydration. Although sampling was conducted on a cool, overcast day with minimal heat stress, we were unable to determine the horses’ water intake, so we cannot completely rule out the possibility of dehydration.
Furthermore, the differences in environmental conditions, management practices, and sampling seasons (e.g., August 2025) between Nakanoshima and Kaimon horses must be considered. Due to geographical constraints, it was difficult to collect samples during the same season; however, the potential influence of seasonal variations on blood biochemical values cannot be entirely ruled out and remains a limitation of this study.
The Nakanoshima horses also showed an elevated GGT level versus the reported value in Kaimon horses [26], suggesting that they are experiencing hepatic dysfunction. Common causes of hepatic dysfunction in horses include ingestion of toxic substances, infectious diseases, and nutritional and metabolic disorders [4]. Examples of toxic substances include poisonous plants containing pyrrolizidine alkaloids (PAs), mycotoxins found in feed [7], and certain pharmaceuticals. PAs are found in many plants around the world. More than 660 PAs and PA N-oxides have been identified in over 6,000 plants mainly contained in the Boraginaceae, Compositae and Leguminosae families [25]. PAs are known to be hepatotoxic, carcinogenic, and genotoxic [5, 12,13,14]. PA-containing plants are thus poisonous to livestock and wildlife and have caused tremendous loss of livestock [10, 22]. There have been several reports of PA-induced hepatic dysfunction in horses [2, 9, 11, 16, 17, 21]. Since the Tokara horses in this study consumed Leopard plant, they may have experienced hepatic damage caused by poisoning from PAs, the toxic compounds found in Leopard plant.
The Leopard plant, which had been consumed by these horses, is an evergreen perennial native to Japan and belongs to the Compositae family; it reportedly contains senkirkine and petasitenine, which are types of PAs known to be hepatotoxic [12, 13]. Horses are known to have developed chronic hepatic damage due to PA poisoning [2, 9, 11, 16, 17, 21]. The liver is the main organ for PA metabolism, although metabolism in other tissues has also been identified. There are three principal pathways for PA metabolism: hydrolysis of PAs to release necines and necic acids; N-oxidation to form PA N-oxides; and oxidation of PAs to produce dehydropyrrolizidine (pyrrolic ester) derivatives [18, 22]. Of the products of these pathways, pyrrolic esters are highly toxic and can cause genotoxicity and carcinogenicity. Accordingly, we consider that the hepatic dysfunction observed in Nakanoshima horses in this study may have been due to PA poisoning resulting from the ingestion of Leopard plant. However, these horses showed no noticeable clinical symptoms and were clinically healthy. This phenomenon may reflect chronic subclinical hepatic dysfunction resulting from the continued intake of small amounts of PAs.
However, in this study, we were unable to detect PA or its metabolites in the blood samples. As PAs are rapidly metabolized after ingestion, they can be difficult to detect in blood. Furthermore, the detection of PA metabolites requires samples to be processed immediately after blood collection [8, 19]; however, such immediate processing was not possible in this study due to logistical constraints. Additionally, as it was not possible to perform a liver biopsy or post-mortem examination for any horse in this study, we were unable to ascertain the presence of findings typically associated with PA poisoning, such as hepatic cell degeneration, necrosis and megalocytosis. To date, there are no reports of direct detection of PAs in horses, and only an isolated report of PA metabolites being detected [16]. Consequently, most reports that have suggested the possibility of PA poisoning are based on clinical signs, blood test results, histological findings, and evidence of plants containing PA in the relevant feed or pasture. Although we were unable to detect PA or its metabolites in this study, we suspected poisoning caused by the consumption of Leopard plant, which the horses were known to be fed on readily. To the best of our knowledge, there have been no previous reports of health problems in horses caused by Leopard plant. To clarify the direct causal relationship, further longitudinal intervention studies—such as monitoring chronological changes in GGT levels after the complete removal of dietary access to Leopard plant—are warranted in cooperation with local caretakers.
Native horses kept in natural environments are believed to avoid eating poisonous plants growing wild in their surroundings; however, we cannot rule out the development of such a preference by the Nakanoshima horses after they became habituated to consuming Leopard plant, initially as human-provided feed, and these horses may then have experienced an adverse health effect as a result of this habitual consumption. These findings suggest that a collaborative assessment of plant species in grazing environments, conducted with daily managers, could support the long-term health of the population. As the Tokara horse is a native breed sometimes utilized as a tourist resource for the purpose of species conservation, care must be taken in managing grazing and foraging areas and in raising awareness amongst travelers and visitors.
Although we suspected liver dysfunction based on elevated GGT levels, reduced genetic diversity is a known issue among Tokara horses [23]. For instance, specific metabolic disorders in other breeds, such as congenital hepatic fibrosis caused by genetic abnormalities, have been previously documented [6, 20]. Therefore, future genetic analyses are warranted to investigate potential links to these disorders.
In conclusion, the results of this study indicate that the Tokara horses on Nakanoshima are clinically healthy; however, the elevated GGT levels observed in blood biochemistry raise the possibility of chronic hepatic damage caused by PA poisoning resulting from the consumption of Leopard plant. Furthermore, the elevated WBC and neutrophil counts suggest possible physiological leukocytosis during sampling, as well as subclinical antigenic stimulation from parasites and chronic localized infections. For the future conservation of Tokara horses and other native equine breeds, it is essential to raise awareness of plants toxic to horses, eradicate these plants from grazing areas and their surroundings, and ensure proper husbandry practices. In addition, care must be taken to prevent the spread of infectious diseases within such populations.
Conflicts of Interest
The authors have no conflict of interest to declare.
Acknowledgments
This study was supported by grants from the Japan Racing Association (JRA), the National Riding Club Association of Japan (NRCA) and Thoroughbred Aftercare and Welfare (TAW). We are grateful to the Toshima Village Office for their cooperation in the collection of blood samples. We are also grateful to Mr. HENRY of Kagoshima University for proofreading this paper.
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