Introduction

Corneal diseases are common in amphibians, and lipid deposition within the cornea is one of the most well-documented ocular diseases of anurans under human care. This condition results when lipid is deposited in the cornea, leading to corneal opacification, decreased visual acuity, and blindness when severe. The condition in amphibians was initially recognized and described in Cuban tree frogs (Osteopilus septentrionalis) in 1986 (Carpenter et al., 1986). It has also been described in a variety of nonamphibian species including dogs, cats, raptors, rabbits, alpacas, giant pandas, and reptiles as well as human and nonhuman primates (Stock et al., 1985; Russell et al., 1990; Shilton et al., 2000; Richter et al., 2006; Holmberg, 2008; Alleaume et al., 2017; Moore and Gjeltema, 2019; Moore et al., 2018; Williams, 2019; Hall et al., 2020; Botello-Bárcenas et al., 2023).

The terms lipid keratopathy or corneal lipidosis are often used interchangeably, but differ in the cause of the lipid deposits. Lipid keratopathy follows corneal neovascularization often secondary to corneal inflammation, whereas corneal lipidosis is usually secondary to corneal degeneration or dystrophy. Lipid keratopathies can occur secondary to ocular or systemic diseases such as trauma, infectious disease, inflammatory conditions, and dyslipidemias, or they may be idiopathic (Stock et al., 1985; Russell et al., 1990; Wright, 2003; Densmore and Green, 2007; Holmberg, 2008; Dubielzig et al., 2010; Williams, 2019; Hall et al., 2020; Boss and Plummer, 2022). Patterns of lipid deposition vary across species and can be multifocal to coalescing, positioned peripherally or axially, and located in the superficial to deep cornea.

Limited research has been performed to determine the pathogenesis of lipid keratopathy in amphibians, and the underlying cause(s) remains unclear. Despite frequent diagnosis in anurans managed under human care, lipid keratopathy is not commonly found in wild populations. This suggests that the artificial housing environments and husbandry practices used for affected amphibians may play an important role in the development of disease (Shilton et al., 2000; Keller and Shilton, 2002; Moore and Gjeltema, 2019; Yaw and Clayton 2019). Homogenous diets with high lipid content, reduced physical activity, and abnormal reproductive patterns that may contribute to frequent or sustained lipid mobilization are frequently encountered in managed collections and have been proposed as risk factors for development of lipid keratopathy in amphibians (Shilton et al., 2000; Shilton et al., 2001; Keller and Shilton, 2002; Wright, 2003; Holmberg, 2008; Pessier, 2018; Moore and Gjeltema, 2019; Kansman et al., 2024).

As lipid keratopathy progresses, the lipid deposits physically obstruct the visual field, leading to partial or complete loss of vision depending on severity (Yaw and Clayton, 2019). For amphibians dependent on sight-triggered feeding and breeding behaviors, the loss of vision associated with the disease can be life-limiting. In addition, the diseased cornea may develop painful secondary conditions such as ulceration and perforation that can severely impact individual animal welfare (Moore and Gjeltema, 2019). Antemortem diagnosis is typically based on ophthalmologic examination findings and exclusion of other causes of corneal opacities (Williams, 2019). Treatment options are limited and often unsuccessful, highlighting the importance of prevention as the primary tool veterinarians have for managing this disease in amphibian collections. As amphibian populations continue to decline worldwide (Griffiths and Pavajeau, 2008; Luedtke et al., 2023), captive breeding programs and optimized care for managed populations are becoming increasingly important to amphibian conservation efforts (Griffiths and Pavajeau, 2008; Silla and Byrne, 2019; Luedtke et al., 2023). Because corneal lipid deposits remain the most commonly reported corneal disease of captive amphibians (Yaw and Clayton, 2019), a better understanding of the risk factors, susceptible species, pathogenesis, and options for prevention and management of this disease are necessary.

Although there are numerous reports of lipid keratopathy occurring in anuran species, reports of the disease in nonanuran amphibians are lacking. The California tiger salamander (CTS; Ambystoma californiense) is a terrestrial urodele amphibian species native to the central valley of California, USA. It is listed as endangered by the U.S. Fish and Wildlife Service and as vulnerable by the International Union for Conservation of Nature (IUCN) (Shaffer et al., 2004; U.S. Fish and Wildlife Service, 2017; IUCN, 2021; U.S. Environmental Protection Agency [U.S. EPA], 2023). These salamanders are opportunistic ambush hunters that rely on eyesight to capture prey (Hattem, 2004; U.S. Fish & Wildlife Service, 2017; U.S. EPA, 2023). This report describes the occurrence of lipid keratopathy in a collection of zoo-housed CTSs.

Materials and Methods

Retrospective review of cases

This project retrospectively evaluated data obtained during physical examinations of salamanders following the Sacramento Zoo Institutional Guidelines for Care and was approved by the Sacramento Zoo Animal Research and Conservation Committee. We adhered to the Association of Zoos and Aquariums code of professional ethics and the American Veterinary Medical Association Principles of veterinary medical ethics.

Electronic medical records and photographs of ocular lesions for each CTS were retrospectively reviewed for the period between 2012 and 2020. Comprehensive ophthalmic examinations were performed for a subset of the animals and included slit-lamp biomicroscopy (Kowa SL15 or SL17 hand-held lamp; Kowa American Corporation, Torrance, CA, USA), rebound tonometry (TonoVet® tonometer, Icare, Vantaa, Finland) in the dog calibration setting, fluorescein stain application (Minims® fluorescein sodium [1% w/v], Bausch & Lomb UK Limited, Kingston-upon-Thames, UK), and indirect ophthalmoscopy with a hand-held 30D lens (Volk Optical Inc., Mentor, OH, USA) when not precluded by corneal opacities. The following data were collected for each salamander: sex, presence or absence of corneal opacities for each eye, tonometry measurements for each eye, estimated mean body weight during the study period, the maximum recorded body weight, and percent weight loss (maximum weight − lowest subsequent weight ÷ maximum weight) for each animal between 2012 and 2020. Lipid keratopathy lesions for each eye were also assigned a severity score of 0–3 based on percentage of cornea affected, with 0 indicating no opacity present, 1 indicating mild disease with <25% of the cornea affected, 2 representing moderate disease with 25–50% of cornea affected, and 3 indicating severe disease with >50% of cornea affected (Fig. 1).

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Results

No ocular abnormalities were observed during physical examinations performed when salamanders arrived at the zoo. Within 3 yr of acquisition, these salamanders reached mature size and by 4 yr (in 2016) many had become overconditioned, with a mean body condition score (BCS) of 5.96 ± 0.84; range 5–8) out of 9 based on visual, radiographic, and ultrasound evaluation of fat stores compared with published references of similar species, because no defined standards exist for CTS (Maerz and Davis, 2007; Jayson et al., 2018).

Corneal opacities were first appreciated in 2016, with 13 of 30 salamanders (43%) having ocular lesions described as bilateral white hazy to cloudy corneal opacities adjacent to the limbus. Because of increased body condition and development of corneal lesions within the group, a diet reduction for weight loss was instituted in early 2017. No other husbandry changes were implemented.

All corneal lesions progressed in their degree of opacity and percentage of cornea affected over the next 4 yr, becoming denser and raised, developing a crystalline-like appearance, and progressing from the periphery toward the axial cornea. The majority of salamanders from this group were sent to other institutions from 2017 to 2019 and were lost to follow-up. Ten remaining salamanders from the group were evaluated in November 2020 by veterinary ophthalmologists and received comprehensive ophthalmic exams. Four of these salamanders were diagnosed with lipid keratopathy lesions based on veterinary ophthalmologist examination findings. The remaining six salamanders had no corneal abnormalities. No salamanders had fluorescein stain uptake at either eye.

Relationships between lipid keratopathy and animal characteristics

There was a high incidence of lipid keratopathy within this group of salamanders, with 13/30 (43%) diagnosed with lipid keratopathy during the 2012–2020 study period.

Sex

All but 2 females (12/14; 86%) developed lipid keratopathy lesions, whereas only 1 male (1/16; 6%) developed lesions. There was a strong positive association between female sex and development of lipid keratopathy lesions (P < 0.0001).

Body weight

The estimated mean adult body weight for affected salamanders exhibiting lipid keratopathy lesions (n = 13) was 64.8 ±7.4 g, with a mean maximal body weight of 76.8 ± 5.7 g. The mean adult body weight for the 17 salamanders without corneal lesions was 62.9 ± 6.0 g, with a mean maximum body weight of 69.3 ± 5.0 g. Salamanders with lipid keratopathy had significantly (P = 0.006) more weight loss after diet reduction was performed in early 2017, with a mean loss of 28.8 ± 7.2% total body weight compared with 20.9 ± 5% total body weight in unaffected salamanders. The one male salamander with lipid keratopathy lesions did not lose weight after dietary change was implemented and was removed from this analysis. Salamanders with lipid keratopathy also had a significantly higher mean maximal body weight than those without corneal lesions (P = 0.001).

Sex vs. weight

The mean adult weight of male salamanders was 62.9 ± 6.0 g, and the mean adult weight of female salamanders was 65.1 ± 7.4 g. There was no statistical difference between sexes for mean adult weight. Females had significantly higher maximal body weight than males (P = 0.001). Females also exhibited a significantly larger degree of weight loss (as a percentage of body weight) than males (P = 0.005).

IOP

The mean tonometry measurement for eyes without lipid keratopathy was 12.9 ± 1.08 mmHg, whereas the mean measurement for eyes with lipid keratopathy (n = 8) was 9.9 ± 2.4 mmHg. There was a significant negative correlation (−0.706) between tonometry measurement and corneal lesion severity score (P = 0.00005; Fig. 2).

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Discussion

Vision is essential for amphibians that are dependent on sight-triggered feeding responses and breeding behaviors, making ocular health critically important when managing these animals under human care. Lipid keratopathy is a well-recognized and important cause of ocular disease and vision loss in amphibians, frequently reported in anurans, but this is the first report of it in a urodele species.

Lipid keratopathy is characterized as white crystalline opacities due to abnormal deposition of lipid within the cornea. Although histopathologic confirmation with oil red O, Sudan black B, and Schultz stain is ideal for definitive diagnosis of small stromal deposits of cholesterol, the gross appearance of hazy white discoloration at the limbus extending over the cornea progressing to a raised surface and intense thick white nodules and plaques on the cornea is considered pathognomonic (Holmberg, 2008; Pessier, 2018; Kansman et al., 2024). A better understanding of potential risk factors for developing lipid keratopathy in amphibians would facilitate earlier detection and could lead to new opportunities for prevention or intervention. The present study describes the occurrence of lipid keratopathy in a group of zoo-housed CTSs and investigated potential relationships between development of lipid keratopathy lesions and factors such as sex, weight, and ocular tonometry measurements.

The ocular lesions in this study were diagnosed by veterinary specialists in ophthalmology and found to be consistent in clinical appearance with corneal lipid deposition as described in other amphibian species (Wright and Whitaker, 2001; Keller and Shilton, 2002; Wright, 2003; Holmberg, 2008; Moore and Gjeltema, 2019; Boss and Plummer, 2022; Kansman et al., 2024). Rebound tonometry values from normal eyes in this study (12.9 ± 1.08 mmHg) were slightly higher than those previously reported for Western tiger salamanders (Ambystoma mavortium) by using similar methodology (11.5 ± 2.7 mmHg) (Cannizzo et al., 2017; Hausmann et al., 2017; Pelych et al., 2019; Balicka et al., 2020; Hausmann et al., 2020; Kansman et al., 2024). This difference may be due to the small sample sizes used in both studies or species differences, or it could be due to inclusion of measurements from diseased as well as normal eyes in the Western tiger salamander study. The results in the present study demonstrate a strong negative correlation between tonometry measurement and the severity of lipid keratopathy lesions, which has not been previously reported in amphibians (Hausmann et al., 2020). This finding may be due to biomechanical changes in the corneal structure that impact tonometry measurements. If this is the case, rebound tonometry measurements may not accurately reflect IOP in affected eyes. Alternatively, lower tonometry readings in eyes with lesions may be due to a true decrease in IOP. Decreased IOP is commonly associated with inflammatory conditions and uveitis. It is possible that lipid keratopathy might induce a reflex uveitis in response to primary corneal disease, or there could be an underlying inflammatory component contributing to or resulting from the corneal lipid deposition. Further study would be necessary to determine the cause of the decreased tonometry measurements found in affected eyes of this study.

Despite lipid keratopathy lesions being one of the most observed ophthalmic diseases in captive amphibians, only limited experimental studies have been performed investigating potential causes. Studies have investigated the relationship of corneal lipid deposits in amphibians with diet, sex, and reproductive cycling (Russell et al., 1990; Shilton et al., 2001; Wright, 2003). Cuban tree frogs have shown increased prevalence of lipid keratopathy in individuals with higher serum total cholesterol and higher ratios of low-density lipoproteins (Shilton et al., 2001). Cuban tree frogs that were fed a high cholesterol diet had increased incidence of corneal lipidosis lesions and higher circulating cholesterol levels than frogs fed a lower cholesterol diet (Russell et al., 1990; Shilton et al., 2001). These studies demonstrate a link between higher circulating cholesterol levels and corneal lipid deposits in amphibians; however, the effect of diet on hypercholesterolemia could not be differentiated from other causes of abnormal lipid metabolism due to the limited scope of these studies (Wright and Whitaker, 2001; Wright, 2003; Moore and Gjeltema, 2019; Williams, 2019). Factors that increase amphibian serum lipid levels should be considered potential contributors for development of lipid keratopathy. Because all salamanders in the present study were managed on the same diet, factors other than diet were considered to be more important contributors to development of disease for this group of salamanders.

The majority of amphibian species prioritize lipid storage over muscle mass once mature body size is reached, and excess dietary carbohydrates and proteins are preferentially converted to fat (Rose and Lewis, 1968; Wright and Whitaker, 2001; Wright, 2003). Amphibians routinely store lipids in large paired or finger-like coelomic fat bodies. Additional lipid storage may occur in inguinal fat bodies, liver, around the heart, in the tail, and in cutaneous or subcutaneous areas to varying degrees dependent on species (Wright, 2003). Coelomic fat bodies are located in close association with the gonad, and their total size has an inverse relationship with developing gonads during times of reproduction (Rose and Lewis, 1968).

Herein, 92% of salamanders with lipid keratopathy lesions were female and 86% of females developed the disease during the study period, whereas only 6% of male salamanders in the group developed lesions. These results are similar to reports in other amphibian species, demonstrating a predilection for disease in females (Shilton et al., 2000; Yaw and Clayton, 2019). In natural settings during reproductive seasons, lipid stores are routinely depleted by reproductive activities. Females require large lipid stores to meet the increased energy demands of vitellogenesis and males for mate-attracting behaviors such as calling, migrating to females or spawning locations, and breeding and courtship displays (Loredo and van Vuren, 1996; Trenham, 2001; Zug, 2001; Wright, 2003). Estrogen promotes increased total plasma cholesterol and triglyceride levels and also stimulates metabolic release of lipid from the fat bodies and liver. As these fat stores are mobilized during normal reproductive stimulation, the systemic free fatty acid concentration markedly increases (Rose and Lewis, 1968). For animals that are not given opportunities for spawning or stimuli for completing the vitellogenesis process, those energy demands do not come to fruition and may lead to prolonged periods of circulating lipids. This could predispose them to abnormal deposition of lipid in tissues, including the cornea (Rose and Lewis, 1968; Russell et al., 1990; Shilton et al., 2001, Williams, 2019). Lack of reproductive opportunity was likely a key contributor to development of obesity, excesses in circulating serum lipids, and development of corneal lipidosis for this group of salamanders. This proposed pathophysiology for development of lipid keratopathy is further supported by the fact that lipid deposition in this group, as well as what is reported in other species, is not generally observed in juveniles and predominantly occurs after animals have reached sexual maturity (Russell et al., 1990; Shilton et al., 2001; Wright, 2003). Because of regulatory permit restrictions that did not allow for breeding within this group of salamanders, the individuals in this study were prevented and discouraged from performing natural reproductive activities. Spawning sites were not provided, salamanders were separated by sex, and environmental cues that stimulate reproductive activity were avoided.

In addition to the profound effect of estrogen stimulation on lipid metabolism, there are also reported differences in the composition of circulating lipids between some male and female amphibians. Female Cuban tree frogs were found to have a significantly higher ratio of very low-density lipid and low-density lipid (LDL) cholesterol over high-density lipid (HDL) cholesterol compared with their male counterparts (Shilton et al., 2001). The cause for female transition of HDL to LDL is unclear, but because elevated LDL cholesterol ratios have been linked to lipid keratopathy in frogs, this difference in lipid composition may be another factor contributing to female predisposition in CTS as well (Shilton et al., 2001). Evaluating cholesterol and lipid levels when performing bloodwork could be helpful in guiding clinical decision-making for this species and warrants further investigation. Unfortunately, cholesterol levels were not available as part of this retrospective analysis.

In the present study, salamanders with lipid keratopathy and females had significantly higher maximal body weights and larger percentages of body weight loss (after an intentional dietary reduction was implemented) than salamanders without lesions and males, respectively. These results make it difficult to evaluate the importance of obesity and weight loss as isolated factors in development of this disease. CTSs are reported to be sexually dimorphic, with males generally being larger than females; however, the females of the current study had significantly higher maximal body weights and larger percentages of body weight loss than males (Loredo and van Vuren, 1996; Trenham, 2001; Hattem, 2004; U.S. Fish and Wildlife Service, 2017; U.S. EPA, 2023). This provides further evidence that in nonbreeding husbandry conditions, female CTSs may be more susceptible to obesity and subsequent development of lipid keratopathy. Given that mobilization of lipids is likely a key contributing factor in disease development, monitoring and management of weight in salamanders under human care to avoid accumulation of large fat stores are appropriate. If weight loss is indicated, slow and gradual weight loss (especially for females) would be recommended. Evaluation of different reproductive management methods as a strategy for preventing obesity and lipid keratopathy would be an important area of future focus that has the potential to positively influence amphibian health and welfare under human care.

Conclusions

This study describes the occurrence of lipid keratopathy in a group of CTSs under human care and evaluated its occurrence for relationships with tonometry measurements, sex, weight, and weight loss. A strong negative correlation was found between lesion severity and tonometry measurements, which has not been previously documented in amphibians. There was also a strong positive association between female sex and development of lipid keratopathy lesions. Females also had significantly higher maximal body weight and higher percentages of weight loss than males. Although diet has historically been implicated as an important factor for development of lipid keratopathy in amphibians, the results of this study suggest that diet alone is unlikely a primary cause, but rather diets causing obesity in combination with reproductive management strategies that affect lipid metabolism may play a more important role in developing disease in CTS under human care and should be a focus of further research. Finding ways to improve our ability to mirror the natural environment as closely as possible for animals in collections under human care appears to be paramount in providing optimum opportunities for health and well-being for these animals.