Stem Cell Therapy in Pinnipedia with Eye Injuries

Stem cell therapy in Pinnipedia with eye injuries

Word count: 9696

Delphine Boone Student number: 01202627

Supervisor: Prof. dr. Catherine Delesalle Supervisor: Dr. Piet De Laender Supervisor: Dr. Constance De Meeûs

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine

Academic year: 2018 - 2019

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis

Preface

With the writing of this word of thanks I put the finishing touches to my thesis after an intensive period of two years. I would like to thank a few people who helped and supported me during this period. To start, Dr. Constance De Meeûs for revising my thesis carefully, always responding fast and enthusiastically and most importantly for giving me great advice. I would like to thank Dr. Piet De Laender for the opportunity to work on this project and help me get in touch with the right people for this thesis. Prof. Dr. Catherine Delesalle for her kind responses and allowing me to work out this topic. Dr. Guy Wouters founder of Fat-Stem Laboratories for providing me freely with stem cell eyedrops. Stephanie Boone, Ellen Verhelst and Carmen Vidal Moreno de Vega for reading the penultimate version.

To all the parks and rehabilitation centres who took the time to fill in my survey, I am very grateful.

To my parents, sister and girlfriend thank you for believing in me even at times where I couldn’t believe in myself.

Table of contents Preface ...... 3 Abstract ...... 5 English ...... 5 Nederlands ...... 5 Introduction ...... 6 Literature ...... 6 Phylogeny ...... 6 Aetiology ...... 6 Epidemiology...... 7 Diagnostics...... 8 Treatment options for eye pathologies ...... 9 Antibiotics ...... 9 Anti-inflammatory therapy ...... 9 Surgical procedures ...... 9 Other ...... 10 Stem cell therapy ...... 10 Research ...... 11 Epidemiology...... 11 Introduction ...... 11 Materials and methods ...... 11 Results...... 11 Discussion ...... 16 Conclusion ...... 17 Stem cell therapy in Pinnipedia ...... 17 Introduction ...... 17 Materials and methods ...... 18 Results...... 19 Discussion ...... 24 Conclusion ...... 24

Abstract

English

Ocular problems are frequently seen in both wild and captive pinnipeds. The aetiology of these eye lesions in wild and captive pinnipeds is very likely to differ.

A survey was made up regarding the epidemiology of ocular problems and sent to parks with captive pinnipeds and rehabilitation centres with wild pinnipeds. The aim of this survey was to get a better insight in these problems since previous literature is either out of date or limited in quantity. Very little is known about stem cell therapy in pinnipeds. However, there is increased evidence in other species that stem cell therapy is beneficial in some eye lesions and is able to regenerate the eye tissue. Stem cells were administered for a period of two weeks to two phoca vitulina (harbor seal) suffering from different eye lesions. These cases have been followed up with photographs. This study may contribute to increasing the knowledge of the epidemiology and aetiology in ocular disorders in pinnipeds and the application of stem cells in these eye problems. Hopefully, new information about these matters will stimulate further research and may ultimately lead to the improvement of animal welfare.

This thesis concludes that the prevalence of eye lesions is much higher in captive pinnipeds in comparison with wild pinnipeds and that eye lesions in captive pinnipeds tend to be rather degenerative whilst in wild pinnipeds this is more of a traumatic origin.

Nederlands

Oogproblemen worden vaak gezien bij zowel wilde als in gevangenschap levende zeeroofdieren (pinnipedia). De etiologie van deze oogafwijkingen bij wilde en in gevangenschap levende pinnipedia is zeer waarschijnlijk verschillend.

Er werd een enquête opgesteld over de epidemiologie van oculaire problemen in pinnipedia. Deze enquête werd naar verschillende parken met in gevangenschap levende zeeroofdieren en revalidatiecentra met wilde zeeroofdieren gestuurd. Het doel van deze enquête was om een beter inzicht te krijgen in deze problematiek, aangezien eerdere literatuur beperkt en/of verouderd is. Over stamceltherapie bij pinnipedia is zeer weinig gekend. Er is echter toegenomen bewijs bij andere diersoorten dat stamceltherapie gunstig is bij sommige oogletsels en in staat is het oogweefsel te regenereren. Stamcellen werden toegediend gedurende een periode van twee weken aan twee phoca vitulina (gewone zeehond) die lijden aan verschillende ooglaesies. Deze twee cases zijn opgevolgd met foto’s. Deze studie kan bijdragen aan de kennis van de epidemiologie en etiologie van oogaandoeningen bij pinnipedia alsook de toepassing van stamcellen bij deze oogproblemen. Hopelijk zal nieuwe informatie verder onderzoek stimuleren en uiteindelijk leiden tot een beter dierenwelzijn.

Deze thesis concludeert dat de prevalentie van oogletsels veel groter is bij in gevangenschap levende pinnipedia in vergelijking met wilde pinnipedia. De oogafwijkingen bij in gevangenschap levende pinnipedia zijn eerder degeneratief, terwijl laesies bij wilde pinnipedia eerder een traumatische oorsprong kennen.

Introduction

This thesis is divided into two parts, being: a literature study and a research part. The literature study will help to get a better insight in eye problems in pinnipeds. Phylogeny, aetiology, epidemiology, diagnostics and treatment options will be discussed. The second topic in this literature review will be about stem cells in general and their possible benefits when administered for eye lesions. The research part will provide a view on epidemiology of eye injuries in captive and semi-wild pinnipeds and will discuss two cases of seals suffering from eye injuries, who received stem cell therapy in both eyes.

Literature

Phylogeny

Pinnipedia is a monophyletic clade containing three extant pinniped families: Otariidae (fur seals and sea lions), Odobenidae (with the walrus being the only non-extinct species) and Phocidae (true seals) (Arnason et al., 2006).

Aetiology

Ocular disorders are frequently seen in both wild and captive pinnipeds (Gerber et al., 1993; Colitz et al., 2010a,b; Miller et al., 2013).

The aetiology of ocular problems is very likely to differ between wild and captive pinnipeds. The predominant cause of ocular disease in captive pinnipeds is the environment. The environment includes water quality and salinity, light intensity, nutrition and spatial characteristics such as the orientation of sun on dry and wet areas, depth, exhibit surface colour (for reflected light) and animal loading per cubic meter (Colitz et al., 2010b).

A study by Colitz et al. (2010b) showed that age, history of fighting, history of ocular disease and an insufficient access to shade are important risk factors in the development of cataract and/or lens luxation in captive pinnipeds. This study focussed on access to shade and showed a 10-fold increased risk to develop these eye problems when there is no access to shade. Also, high UV exposure times are an important risk factor for developing ocular disorders in captive pinnipeds. For example, reflective, lightly coloured pools and situations that force the animals to look directly in the sun predispose these animals to ocular disorders (Gage, 2011).

Water quality parameters also have a role in the development of ocular diseases. De Haan (2008) found that chlorine significantly increased (P < 0.01) the size of corneal lesions in captive Zalophus californianus (California sea lions). To purify and disinfect the water, filter systems and chemical products such as chlorine are often used. When chlorine is added to water that contains organic material (mainly ammonia), it forms chloramines. These chloramines cause irritation and damage the eyes (De Haan, 2008); Gage (2011) observed epiphora and blepharospasm in pinnipeds which were exposed to residual ozone. Ozone is a strong oxidative which is widely used as disinfectant for marine mammal life support systems which are essential to maintain clean water (Gage, 2006; Gage, 2011). Marine mammals are maintained in fresh waters, brines (sodium chloride dissolved in tap water), artificial sea water (with some or all of the major ions dissolved in tap water) or sea water (which sometimes is diluted with fresh water and therefore becomes brackish) (Spotte, 1991). Fresh water

contains a lower dose of salt and other ions and increases the chance of eye lesions in comparison with animals living in the other types of water (Spotte, 1991; Dunn et al., 1996). Dunn et al. (1996) found a significant correlation (P < 0.01) between harbor seals living in fresh water and the development of cataract. A three times higher prevalence of cataracts occurred in fresh water holding facilities than in salt water; In addition, Dunn et al. (1996) found a strong association (P< 0.00001) between California sea lions held in fresh water and corneal oedema.

Genetic predisposition is also a risk factor for developing eye problems, however more research is needed to underpin which genes are involved in eye pathologies in pinnipeds (Colitz et al., 2010b).

Epidemiology

Eye injuries can be seen in both wild and captive pinnipeds (Gerber et al., 1993; Colitz et al., 2010a,b; Miller et al., 2013), however the prevalence is higher in the captive animals (Dunn et al., 1996).

Greenwood (1985) examined 126 captive pinnipeds of 7 species (Otaria flavescens (South American sea lion), California sea lion, Arctocephalus pusillus (brown fur seal), Arctocephalus gazella (Southern fur seal), harbor seal, Halichoerus grypus (grey seal) and Mirounga leonina (Southern elephant seal)) in 26 European zoos and 28 (22%) of them had an eye lesion. In this study many more otariids than phocids were examined, however the highest prevalence of ocular disease was in phocids. 8 of 25 phocids (32%) were affected, compared with 20 of 101 otariids (19,8%). The ocular disorders that are most frequently found in captive pinnipeds are: corneal oedema, corneal opacities, corneal lacerations, chronic keratitis, cataract, lens luxation and blepharospasm (Gage, 2009; Gage, 2011; Miller et al. 2013).

A study by Colitz et al. (2010a) evaluated the eyes of 113 captive otariids (100 California sea lions, 6 Eumetopias jubatus (Stellar sea lions), 5 brown fur seals, 1 Arctocephalus townsendi (Guadalupe fur seal) and 1 Callorhinus ursinus (northern fur seal). 73 out of the 113 animals (64,6%) and 142 eyes in total (62,8%) were affected with keratitis. Keratitis stage 1 (initial stage characterised by a superficial grey-white corneal opacity) was found in 78 of 226 eyes (34,5%) in 37 animals (32,7%) with an average age of 14,17 years. Keratitis stage 2 (affects 10-20% of the cornea with ulceration, secondary infections and oedema) was seen in 30 eyes (13,3%) in 16 animals (14,2%) with an average age of 19,43 years and stage 3 keratitis (affects 20-80% of the cornea with recurrent ulcerations, secondary infections and/or abscesses and oedema) was seen in 34 eyes (15%) in 20 animals (17,7%) with an average age of 19,22 years (P= 0.0077). Groups were made according to age (0-5 years: n=40, 6-10 years: n= 32, 11-15 years: n= 26, 16-20 years: n =64, 21-25 years: n=56, 26-30 years: n=6, >30 years: n=2). All groups of animals older than 5 years had a 50% greater incidence of keratitis. Animals over 26 years (n=8) had an incidence of 100%. In another study by Colitz et al. (2010b), eyes from 111 captive pinnipeds (99 California sea lions, 10 harbor seals and 2 Odobenus rosmarus (walruses) from 9 facilities in the United States and the Bahamas were examined. In this study pinnipeds younger than 15 years old were significantly (P < 0.001) less susceptible to develop cataracts and/or lens luxation in comparison to pinnipeds older than 15 years old.

Gerber et al. (1993) studied 1446 pinnipeds of 6 species (California sea lion, Mirounga angustirostris (northern elephant seal), harbor seal, northern fur seal, stellar sea lion and Guadalupe fur seal) stranded along the central and northern California coast. These animals were hospitalized and released to the wild after a successful medical treatment. Eye lesions that could be seen were blindness, corneal

ulcer, corneal oedema, hypopyon, conjunctivitis, cataract, trauma and puncture wounds. In some cases, blindness was a transitory problem, which disappeared once the body condition improved. 8,5% of a group of California sea lions (n=765), 7% of admitted harbour seal pups (n=227), 21% of northern elephant seal pups (n= 225) were diagnosed with an eye lesion. During the study period 154 (37%) elephant seals were admitted with a skin disease of unknown origin, 5% of them also had an ocular problem.

Diagnostics

Before beginning the eye examination, evaluating the animal from a few feet away is advised. Pay attention for facial symmetry, globe and eyelid position, altered head position, blepharospasm, eyelid abnormalities or accumulation of discharge around the eyes. It is recommended to examine the animal from different angles since it can be difficult to detect subtle changes such as mild exophthalmos (Welch, 2007).

A focal light source, a direct ophthalmoscope and a 20 or 30 dioptre (D) condensing lens are the minimum of equipment necessary in a veterinarian practice. Schirmer tear test (STT) strips as well as fluorescein strips or fluorescein solutions in a vial for single use should also be available (Heinrich, 2014).

The ophthalmic exam should be performed in a consistent and organized manner so that a routine can be developed. Ophthalmic examination in general consists of evaluating the pupil and eyelids, controlling the cornea for oedema, vascularisation and opacities, control of the anterior eye chamber for depth and clarity and checking the transparency of the lens (Welch, 2007).

Microbiology samples can be obtained with sterile swabs. Samples for cytology can be collected by a direct impression smear, cytobrushes or with the blunt end of a scalpel blade. These last two are especially helpful when collecting samples from the corneal surface. Samples should be collected before applicating a topical agent since these may interfere with the laboratory results (Heinrich, 2014).

Fluorescein is widely used to detect cornea ulcerations and control the nasolacrimal passage. Superficial lesions of the cornea will stain slightly less intense than stromal lesions. In case of using fluorescein-impregnated paper strips it is important to first wet these with a drop of saline before carefully applicating them to the conjunctiva (Heinrich, 2014).

A STT is typically used in dogs to diagnose keratoconjunctivitis sicca (Heinrich,2014).

Ophthalmic examination in pinnipeds is challenging for different reasons: the presence of a prominent nictitating membrane, strong eyelids, the ability of these animals to retract the globe into the ocular cavity, very small pupils that interfere with a clear visualization of the lens and the retina and, lastly, when administering topical mydriatics on the cornea, pinnipeds tend to not dilute their pupils very well (Gulland, Haulena and Dierauf, 2001)

Different bacteria from traumatic eye lesions, conjunctivitis and keratitis have been cultured in pinnipeds (Gulland, Haulena and Dierauf, 2001).

Visually impaired pinnipeds tend to exaggerate the use of their vibrissae (whiskers) when investigating noises or new surroundings and extend them for a prolonged time in comparison with not visually impaired pinnipeds. Vibrissae are very sensitive to movement and therefore make it difficult to evaluate the menace response in pinnipeds. Visually impaired pinnipeds placed in new surroundings may not avoid obstacles as good as not visually impaired pinnipeds, however they can adapt very quickly using tactile and acoustic signals which makes it hard to diagnose blindness in these animals (Gulland, Haulena and Dierauf, 2001).

Treatment options for eye pathologies

Antibiotics In case of a bacterial infection or a secondary bacterial overgrowth, antibiotics can be used. Antibiotics should be used based on sensitivity results. Neomycin (an aminoglycoside) is the first choice for treatment of corneal ulcers caused by Pseudomonas aeruginosa. Gentamycin (an aminoglycoside) is often used in the treatment of melting ulcers caused by P. aeruginosa. Ofloxacin (a fluoroquinolone) can also be used for this indication (Barnett, 1998).

Keratitis in combination with a superficial ulcer should be treated immediately and aggressively, as loss of the upper epithelial layer predisposes to secondary infections. When a secondary infection is diagnosed antibiotic and anti-inflammatory therapy should be started. Initially a topical antibiotic should address the possible pathogens in the water. Oral medication, typically doxycycline (a tetracycline) is often used since this antibiotic stabilises corneal stroma, accelerates epithelialization and has anti-inflammatory properties (Colitz et al., 2010a).

Anti-inflammatory therapy Eye lesions are very painful. An oral nonsteroidal anti-inflammatory drug (NSAID) is administered against pain and secondary uveitis (Colitz et al., 2010a). NSAIDs such as meloxicam and carprofen can be given orally to reduce pain and inflammation in patients who are suffering from painful ocular diseases such as keratitis and lens luxations.

Corticosteroids such as dexamethasone are used topically by some veterinarians to reduce scarring (Barnett, 1998). But care has to be taken when using corticosteroids because they decrease fibroblastic activity and inhibit wound healing, which could result in a cornea perforation (Waring, 1979).

Surgical procedures Lensectomy is a technique by which the lens is cut and aspirated through a bore needle (Kanski and Crick, 1977). This technique has been applied with success in pinnipeds, resulting in an improved vision and reduced pain and it has been shown that surgery prevents secondary eye problems (Higgins and Hendrickson, 2013). Phacoemulsification is a technique in which the lens gets ultrasonically fragmented and emulsified before being aspirated from the eye (Brooks et al., 2014). This technique is commonly used for cataract treatment in domestic species, however it is ineffective in adult pinnipeds since the round and dense lens is too difficult to break down and remove by phacoemulsification. It can therefore only be applied successfully in young pinnipeds (Higgins and Hendrickson, 2013).

Other Topical cyclosporine (2%) or tacrolimus (0,03%) can be used to reduce the recurrence of keratitis (Colitz et al., 2010a). Both products stimulate tear production and suppress the immune system1. Vitamin A can be added to achieve a better epithelisation. Carbonic anhydrase inhibitors such as brinzolamide eye drops reduces pressure within the eye and can be used against glaucoma2.

Stem cell therapy

In the last 20 years, stem cell research emerged, resulting in a better understanding of its characteristics and its therapeutic potential (Markoski, 2016).

Stem cells are defined by four criteria. First, they should have the ability to regenerate through mitotic division (undergo self-renewal), in order to sustain the population. Secondly, a single stem cell-derived daughter cell must be able to differentiate into more than one cell type, an example are hematopoietic stem cells that give rise to all hematopoietic cells. A third criterion is that stem cells must be able to functionally repopulate the tissue of origin when transplanted into this damaged tissue. The last criterion is that stem cells should give rise to differentiated descendants even in the absence of damaged tissue (Verfaillie et al., 2002).

Stem cells can broadly be classified as embryonic or adult, depending on the developmental stage they were obtained from. There are different populations of stem cells, all of which have other properties. Starting from totipotent stem cells that make it possible to create a completely new individual, this property is retained by the zygote and its early progeny up to the 8-cell stage of the morula (Fortier, 2005). This cell is capable to form trophoblast cells, in addition to cells of the mesoderm, endoderm or ectoderm, which are necessary for the implantation of the developing embryo (Van Haver et al., 2008). Pluripotent stem cells, originating from the ‘inner cell mass’ of the blastocyst, can differentiate into cells of the endoderm, ectoderm or mesoderm (Van Haver et al., 2008). However, they can’t produce a new individual on their own (Verfaillie et al., 2002). Multipotent stem cells isolated from adult organs can differentiate into different organ-specific cell types. They yield a more restricted subset of cell lineages (Fortier, 2005; Van Haver et al., 2008). Progenitor cells are undifferentiated cells that are capable of forming one particular, differentiated cell type (Van Haver et al., 2008). They are therefore not seen as true stem cells since they are more or less committed to becoming cell types of a particular tissue (Li et al., 2014).

Dysregulation of embryonic stem cells can, in vivo, cause a teratoma (which is a benign tumour composed of a disorganized mixture of tissue, composed by cells of all three embryonic germ-layers (Wesselschmidt, 2011)) and there’s evidence that they are responsible for genome instability in vitro. This makes the use of embryonic stem cells in veterinary medicine still controversial (Markoski, 2016).

Mesenchymal stem cells are a pool of self-stem cells originating from the mesoderm and able to differentiate through a series of separate and unique lineage transitions into a variety of end stage phenotypes (bone (osteoblast), cartilage (chondrocyte), tendon, ligament, marrow stroma, adipocyte, dermis, muscle and connective tissue). The primary function of these adult stem cells is physiological

1 Package leaflets archived from https://www.diergeneeskunde.nl/media/filebank/772062e077b04792bfa19f9f2e5108cb/dgk_apotheek_ bijsluiter_ciclosporine-2-oogdruppels.pdf and https://www.diergeneeskunde.nl/media/filebank/6fbb8359d0524693b2d6a6f3d972d857/dgk_apotheek _bijsluiter_tacrolimus-oogdruppels.pdf on 3 May 2018. 2 Package leaflet archived from https://www.medicines.org.uk/emc/files/pil.3819.pdf on 3 May 2018.

cell renewal. In pathological situations, such as inflammation, trauma or ischemia, they are able to substitute dead cells. Mesenchymal stem cells have the best chance of therapeutic success since they are able to modulate the immune system, activate homing factors and allow cells to enter the inflammation site, which is beneficial for tissue repair. They are typically isolated from bone marrow or adipose tissue (Caplan, 1991; Markoski, 2016).

Stem cell therapy can be used to repair damaged tissues caused by oxygen deprivation at cellular level and therefore may be useful for ischemic retinopathies (Van Haver et al., 2008).

Furthermore, stem cell therapy has been used in the treatment for degenerative ocular diseases, to replace lost neurons, restoring neural pathways or as paracrine mediated therapy. In these paracrine mediated therapies, stem cell-derived trophic factors protect compromised endogenous retinal neurons from dying and induce the growth of new connections. Mesenchymal stem cells may be an eligible source of paracrine factors preventing retinal ganglion cells from dying and stimulate the regeneration of the axons in the optic nerve in degenerative ocular disease (Mead et al., 2018).

The main issue for a successful stem cell therapy when injected intravenously is that the transplanted cells need to find their way to the damaged tissue. When they fail to find their way, the cells die in the blood circulation or they get trapped in other organs. The best procedure is to inject stem cells directly into the damaged tissue (Naderi-Meshkin et al., 2015).

Research

Epidemiology

Introduction Most of the data found in literature is either out of date or limited in quantity. This previous literature (Gerber et al., 1993; Colitz et al., 2010a,b; Miller et al., 2013) suggests ocular problems to be of frequent occurrence in pinnipeds. The aim of this study was to have a better view on the prevalence of eye lesions in both wild (rehabilitating) and captive pinnipeds and to gain knowledge about the lesions that are most common.

Materials and methods A short survey (appendix I) was drawn up in three languages (Dutch, English and French) regarding eye problems in pinnipeds. The survey was sent by e-mail across Europe, North America, South America and Australia to both rehabilitation centres with wild pinnipeds and parks with pinnipeds in captivity. Pinnipeds in rehabilitation centres were classified as wild pinnipeds since they are typically stranded due to different causes and are released back into the wild after rehabilitating. A population of pinnipeds in captivity were present as well in this study, these animals were classified as captive pinnipeds. This separation was included because it can be assumed that pinnipeds in rehabilitation centres are closer to wild pinnipeds than the ones living in captivity.

Results In total 258 pinnipeds were part of this study of which 234 wild pinnipeds from 7 different rehabilitation centre and 24 captive pinnipeds from 3 different parks. A division was made in the results, analysing pinnipeds in rehabilitation centres and captive pinnipeds in parks separately (graph 1 and 2) and in proportion (graph 3). Extra valuable information regarding the housing and the

behaviour of the animals from the different parks and rehabilitation centres is added to give an overall better image.

Around 6,4% of the pinnipeds in rehabilitation centres showed eye lesions. Most seen ocular problems were corneal ulceration and oedema. Perforated corneas accompanied by one sided blindness and one case of chronic bilateral keratitis that evolved in a bilateral ulceration were seen as well. The cause for these eye lesions were either traumatic or unknown. There was one unclear case of mature cataract possibly accompanied by lens coloboma. This could possibly be a degenerative or congenital lesion.

Of the 24 pinnipeds in captivity, half showed ocular problems. The ocular problems seen in these animals were mostly degenerative. Lens luxations (33%), cataracts (25%), keratitis (25%) and corneal ulcerations (17%) were the lesions that were present the most. One animal was completely blind due to a headshot and therefore ended up in captivity. Another animal had an eye infection.

OCULAR PROBLEMS IN OCULAR PROBLEMS IN PINNIPEDS IN CAPTIVE PINNIPEDS REHABILITATION CENTRES

Present 6,4% Present Present Absent Present 50% 50% Absent Absent

Absent 93,6%

Graph 1: the prevalence of eye lesions in wild Graph 2: prevalence of eye lesions in captive (rehabilitated) pinnipeds, with n=234 pinnipeds, with n=24

COMPARISON OF THE APPEARANCE OF EYE LESIONS IN CAPTIVE AND REHABILITATING SEALS

Wild 11,4% Wild Captive Captive 88,6%

Graph 3: comparison of the appearance of eye lesions in captive pinnipeds (50%) and wild pinnipeds in rehabilitation centre (6,4%)

“Rehabilitation centre 1”: In March 2018, 22 pinnipeds were being taken care of in the centre. One of them, a juvenile grey seal, had a traumatic eye lesion. The lesion included corneal oedema and corneal ulceration. Corneal ulcerations are mostly seen due to trauma in this rehabilitation centre. Occasionally one-sided juvenile cataract is seen as well. It is still unclear whether this condition is congenital or is secondary after trauma.

This centre was contacted again in November 2018, at that time 21 pinnipeds were present in the centre. Four of them had ocular problems. One individual had a cornea defect. This was without complaints. Another individual had a perforated cornea due to a traumatic injury thus an enucleation of the eye was performed. The third individual had a corneal ulceration. The fourth individual (figure 1) had an unclear condition, with differential diagnoses: mature cataract, lens coloboma (an anomaly of the lens shape) and microphakia (an abnormal smallness of the lens).

Figure 1: unclear case but highly suspected of mature cataract and lens coloboma. Picture by Drs. M. Geut, A Seal Stellendam

“Rehabilitation centre 2”: In February 2018, 24 pinnipeds were present in rehabilitation centre 2, all of them were California sea lions. Five of them suffered from eye injuries. One was shot in the head and lost an eye as a result. A second individual developed an ulcer during rehabilitation with subsequent scarring and loss of vision in that eye. A third individual came in with a ruptured globe due to unknown causes and the eye was enucleated. Two others came in with unilateral corneal oedema of which the cause was unknown. In this rehabilitation centre eye injuries are not uncommon and occur due to natural causes as well as intraspecific competition between animals in the facility during rehabilitation.

“Rehabilitation centre 3”:

In December 2017 this centre housed 65 pinnipeds, of which one, a harbor seal, had a traumatic eye injury (figure 2).

This centre was contacted again in March 2019, this time the centre housed 71 pinnipeds, of which one male animal had a corneal ulceration of unknown origin.

Figure 2: traumatic eye lesion, picture by Veterinary Department Sealcentre Pieterburen

“Rehabilitation centre 4”:

In October 2018, 24 pinnipeds were present at the facility of which two seals had eye injuries. One harbor seal’s eye was damaged by physical injury, most likely done by a seagull (figure 3). There was no infection present and the eye is now healed. However, the individual is blind in that eye. Another grey seal has a punctured eye. Both seals were released back in the wild approximately a month later.

The centre mainly sees eye problems in males. Eye injuries are presented equally in the different species of seals that they care for. Eye infections in harbor seals have arisen due to the phocid herpes virus-1 (PhHV-1). In Figure 3: traumatic eye this centre, 2 seals presented corneal oedema in the past two years and lesion in the left eye, picture both of them were the result of physical injury. The swelling caused a by Seal Rescue Ireland lot of discomfort to the seals and, therefore, resulted in full enucleation of the eye. Seals with one eye, or unilateral view, are still able to hunt as good as seals with two complete functioning eyes. Corneal ulcerations in seals were seen in 5 cases over the past two years.

“Rehabilitation centre 5”: In October 2018 this centre housed 4 pinnipeds. One of them, a 2-year old female California sea lion had a chronic bilateral keratitis that evolved in a bilateral corneal fibrosis and finally a bilateral ulceration of unknown cause. In a blood analysis she was positive for leptospirosis.

“Rehabilitation centre 6”: In April 2018, three grey seal pups were presented at this centre. None of them had eye lesions.

“Park 1”: This park owns 4 harbor seals and 4 California sea lions. There is one 32-year old seal that suffers from both-sided senile cataract and has a lens luxation in his left eye. His son (18 years old) has what seems like an early stage of cataract with subtle white discoloration. Two sea lion brothers (5 and 6 years old) show a keratitis stage 1 (following the characterization of progressive keratitis in Otariids by Colitz et al., 2010). Keratitis is typically seen during periods of stress (for example moving an animal in or out of the group), this has made it possible for the caretakers of this park to take medicinal preventive actions. Keratitis is possible to have a genetic predisposition however, in this case, both parents of these two brothers didn’t show any symptoms of keratitis. The remaining two seals without eye lesions are 16 and 10 years old. Furthermore, there were two sea lions aged 2 and 5 without ocular problems.

Figure 4: Characterization of progressive keratitis in Otariids (Colitz et al, 2010)

Figure 5: both-sided senile Figure 6: early stage of cataract with cataract and lens luxation in the subtle white discoloration, picture by left eye, picture by Delphine Delphine Boone Boone

The 32-year old seal that suffers from both-sided senile cataract and a lens luxation in his left eye, gets 2 mg/kg bodyweight Rimifin (caprofen, against pain), two Zantac tablets (75 mg/tablet ranitidine, stomach protectors), Cavasan eyedrops (chloramfenicol and vitamin A) four times a day in both eyes and Azopt (lowers eye pressure) three times a day in the eye which has the lens luxation. For two years now, he has a covered place with shade which he can enter freely or where his caretakers put him when the sun is shining too bright. The two sea lions only get medication when the keratitis recurs. When this happens, they receive meloxicam (15 mg/ml, against pain) once a day with a syringe corresponding to the number of kilograms they weigh. Doxycycline 10 mg/kg bodyweight is administered once a day (or divided over two feedings) and Cavasan four to six times a day in the troubling eye. Animals are kept out of the water for at least 5 minutes after administering medication.

Water quality

The water, in which the animals live here, originates directly out of the sea. After it has undergone filtration, it is led into the tank. There is no chlorine in the water. In order to prevent reflection of light in the eyes, algae are present in the tank.

“Park 2”:

This park has 4 California sea lions and 6 harbor seals. There’s one 28-year old sea lion with lens luxation in her right eye. The other sea lions are 19, 14 and 11 years old and show no ocular problems. 5 out of 6 seals have eye problems: two of them have a lens luxation and severe keratitis (both aged 29, one showed unilateral keratitis, the other one bilateral), one has cataract (age 17) and two have corneal ulceration (age 12 and 29). The youngest seal (age 10) doesn’t have any eye problems. The 29-year-old seal with unilateral lens luxation is the mother

Figure 7: lens luxation in the right eye of the 12-year-old seal with corneal ulceration. The 29-year-old of a Californian sea lion, picture by seal with corneal ulceration is the mother of the youngest seal Delphine Boone without ocular problems.

Water quality

The animals live in salt water. The water is filtered by a filter with fibre glass. From March to September a small amount of chlorine is added to the water which serves as a disinfectant. In the period from September to March no chlorine is used in the basin. The water is treated with a chlorine shock every 6 weeks. The animals do not have access to this basin during this chlorine shock.

“Park 3”: This park houses 2 California sea lions, 3 harbor seals and a grey seal. The grey seal lost his eyes when he was a pup stranded on a beach in the UK. He got shot with a gun in the head, survived, but lost both of his eyes. He is guessed to be approximately 11 years old. One harbour seal of 21 years old has an eye infection. The other two seals are age 17 and 15. The two sea lions are 17 years old.

Water quality

The tanks of these animals are filled with sea water. Sand filters, UV sterilization and a copper system against algae are being used by this park.

Discussion In comparison with the findings of Gerber et al. (1993), who studied the eyes of pinnipeds stranded along the central and northern California coast: conjunctivitis, hypopyon and cataract were not described, with exception of one unclear case with a possible mature cataract. Note that conjunctivitis and hypopyon were not included in the survey for this study, however cataract was. Percentages of eye diseases in the stranded pinnipeds by Gerber et al. (1993) (7% of harbour seal pups and 8,5% of California sea lions) were comparable with pinnipeds in rehabilitation centres from this study (6,4%), with exception of the northern elephant seal pups (21% eye lesions) and skin-diseased elephants seals (5% eye lesions) who were not present in this study.

All of the captive pinnipeds with a lens luxation had were of old age, varying from age 28 to 32. Cataract was present in three captive animals with age 17 or older. Younger animals showed fewer ocular problems in comparison with the older pinnipeds in captivity. This corresponds with the findings of Colitz et al. (2010b). However, prevalence’s show some differences in both studies. In this study 4,2% of captive pinnipeds showed a dual diagnosis of cataract and lens luxation, in comparison with the study by Colitz et al. (2010b) that showed a 15,3% prevalence. Furthermore, in this study 8% of pinnipeds were diagnosed with cataracts alone whilst in the study by Colitz et al. (2010b) this was 34,2%. Of the 48 eyes evaluated in this study 20% had lens luxation, cataracts or both; In the study by Colitz et al. (2010b) of the 222 evaluated eyes 46,8% had lens luxation, cataracts, or both. Differences might be explained due to differences in sample size, localisation of the parks and species of pinnipeds. This study included 24 pinnipeds, of which 13 harbor seals, 10 California sea lions and 1 grey seal from 3 facilities located in Western Europe. The study by Colitz et al. (2010b) included 111 captive pinnipeds, of which 99 California sea lions, 10 harbor seals and 2 walruses from 9 facilities located in the United States or the Bahamas.

The eye lesions in wild pinnipeds seem to be of a more traumatic origin in comparison with eye lesions in captive pinnipeds, which tend to be rather degenerative.

Of the 44 contacted facilities only a small amount responded and/or participated in this study. Of the 37 contacted parks, 3 participated (8,1% response). All but one of the 7 contacted rehabilitation centres filled in the survey (85% response). This difference in response raises the question: “Why is

there such a low response rate from parks with captive pinnipeds?”. Do rehabilitation centres have more volunteers and thus more time? Or are they just more invested in their animals? Is there a lack of time due to a high work pressure in parks with captive pinnipeds? Are parks scared that information may be used against them by animal right activists and therefore influence the public opinion which may result in less profit? Could adjustments in the structure of the survey have led to more responses? Or is the difference in response just a coincidence? All these questions aside, the lack of responses made the sample size for captive pinnipeds too small to form a strong conclusion. Moreover, drawing conclusions about genetic and gender predisposition for eye lesions was made impossible due to the same reason.

As mentioned earlier, a division between pinnipeds in captivity and “wild” pinnipeds in rehabilitation centres was made. It may be interesting if future research could examine three groups, being: captive pinnipeds, pinnipeds in rehabilitation centres and wild pinnipeds. A possibility could be to let researchers dive underwater and examine the eyes of pinnipeds to get a full image of eye problems in these animals, however, it seems difficult to implement this plan in reality.

Conclusion This study agrees with earlier literature (Dunn et al., 1996) that ocular problems occur more in captive pinnipeds in comparison to wild pinnipeds. However, it is important to keep in mind that animals in captivity tend to become older then wild pinnipeds and some eye problems seem to be age related. While the eye lesions in wild pinnipeds seem to be of a more traumatic origin, lesions in captive pinnipeds tend to be rather degenerative.

Stem cell therapy in Pinnipedia

Introduction Up until now, very little is known about stem cell therapy in pinnipeds. However, there is increased evidence in other species that stem cell therapy is beneficial in some eye lesions and is able to regenerate the eye tissue (Van Haver et al., 2008; Mead et al., 2018).

Based on a study of Demirayak et al. (2016) it is considered that allogeneic stem cells can be precursors for stromal repopulation, which will improve the regeneration capacity of the corneal stroma. In this study, they have tested different pools of stem cells to regenerate eye lesions in rats. A surgical penetrating eye lesion was made in 3 groups of rats. Bone-marrow-derived mesenchymal stem cells were tested in one group of rats (n=15), adipose-tissue-derived mesenchymal stem cells were used in a second group (n = 15) and the last group was the control group (n=10). The mean keratocyte density (both anterior and posterior) was significantly increased in the first two groups compared to the control group. The expression of aldehyde dehydrogenase (ALDH), CD34 and the corneal stroma- specific proteoglycan keratin by the applied stem cells was studied. All three factors were significantly increased, which proves that the stem cells have differentiated into functional keratocytes. Therefore, this study suggests that bone-marrow-derived stem cells and adipose-tissue-derived stem cells may be used as treatment in eye pathologies causing keratocyte loss, such as traumatic loss of keratocytes and corneal lesions induced by infections. Bone marrow mesenchymal stem cells (BM-MSC) and adipose tissue-derived mesenchymal stem cells (AT-MSC) showed a similar migration to the stroma, viability and differentiation into efficacious keratocytes after injection in the anterior chamber of the cornea

through the penetrating wound. However, AT-MSC are preferred since they can be isolated in large numbers rather easily and can be obtained more safely in comparison with BM-MSC.

The best procedure for successful stem cell therapy is to inject stem cells directly into the damaged tissue (Naderi-Meshkin et al., 2015).

Materials and methods As a second part of this thesis, stem cell therapy has been performed in two captive harbor seals for a period of two weeks. The animals, one male and one female, are both age 29 and live in the same park.

The water in which the animals live is filtered with use of fiberglass (AFM). It is a closed filter system. The filter itself is cleaned twice a week. Chlorine, pH values and salt levels are controlled by the caretakers on a daily basis. The animals live in salt water. Salt is added manually to a salinity of 22 ‰. During the warmer months (March - September) a small amount of chlorine is added as a disinfectant to the water. The concentration of chlorine and the pH are monitored by a Depolox system. Values of free chlorine range from a minimum of 0,4 mg/l to a maximum of 0,6 mg/l, combined chlorine should always be below 0,5 mg/l and the pH between 7,2 and 7,6. In the colder months (September - March) no chlorine is used in the basin. The water is treated with a chlorine shock every 6 weeks. This is a procedure that is done to convert chloramines (combined chlorine) back in free chlorine. The target of this chlorine shock is to destroy bacteria and other microorganisms that may have some resistance against the normal values of chlorine, prevent growing of algae and destroy chloramines, which are responsible for the typical chlorine smell and irritation of the eyes. The animals do not have access to their basin during this chlorine shock. The seal basin can be split in two smaller basins (both with their own filtration installation) by a watertight bulkhead. Before the chlorine shock and splitting the basin, all animals are asked to come to one side to make sure that that the animals no longer come in contact with the water in the other basin. A fast dissolving disinfection product is being used, such as Melpool 70/G or 63/G. The animals are then asked to come to the 'fresh' basin and the accommodation is split so that the other side can also be shocked.

Both animals tested positive on a fluorescein eye test. Only animals with a positive fluorescein test were included, to make sure stem cells can penetrate into the damaged tissue. The two seals both received six eyedrop of stem cells (appendix II) in each eye, twice a day for two weeks. Pinnipeds should be trained to get eyedrops. If not, the animals will close their eyes firmly after the first administration of the eye drops. The chosen animals were trained so this was not a problem. After application the animals were held out of the water for one minute, so the stem cells didn’t flush away. Pictures have been taken to follow-up the healing process.

Trafloxal (Ofloxacine), one drop twice a day, is administered in the affected eye of animals in this park with a positive fluorescein test thus indicating a cornea defect. Animals frequently pinching or closing their eye(s) receive meloxicam (NSAID) orally.

During the application of stem cells, no other medication was administered in the two seals.

Results Case 1: a male harbour seal, age 29, suffers from a bilateral lens luxation diagnosed in June 2017. Macrophtalmia and exophthalmia are clearly visible in the right eye of this seal. The diffuse vascular congested sclera is a clear sign of inflammation. Scleral vascular injections can be observed as well. The condition has evolved chronically with permanent changes in the right eye structure and peri- orbital structures. In the left eye a severe chronic superficial keratitis (pannus), with a lot of pigmentation as well as ingrowth of blood vessels can be seen.

Before becoming chronic, the scleral vascular injections were treated topically with diclofenac (NSAID).

16/01/2018: 12/04/2018:

4/06/2018:

16/07/2018:

A fluorescein test was performed and was positive for the left eye. Two central erosions or ulcerations of the cornea were made visible.

26/07/2018:

Beginning of the stem cell therapy. Both eyes were treated however we expected potential results more in the left eye since that one was fluorescent positive.

09/08/2018:

Stem cell therapy was stopped. No changes in the eye could be observed.

25/08/2018:

Case 2: a female harbour seal, age 29, suffers from a lens luxation in the right eye and superficial keratitis in the left eye that was first noticed in December 2017.

08/01/2018:

Severe chronic superficial keratitis (Pannus) is seen in the left eye. Corticosteroids were administered to slow down the process and stimulate remodeling of the tissue.

16/01/2018: 12/04/2018:

04/06/2018:

The right eye has a blue appearance, which is suggestive for glaucoma (possibly secondary to the lens luxation) or cataract. The left eye appears enlarged and structureless. This is the result of a severe keratitis.

10/07/2018:

Strong hypertrophic sclera of the left eye is seen. The image seems to be similar to plasma cell conjunctivitis, a condition in which the third eyelid becomes inflamed due to accumulation of plasma cells (inflammatory cells). Plasma cell conjunctivitis leads to discoloration and thickening of the third eyelid and can spread to the other mucous membranes of the eye. Aetiology of this condition is an inadequate reaction of the immune system, which is exacerbated under the influence of UV light. The condition is mainly seen in German Shepherd Dogs and often occurs at the same time as keratitis

(pannus)3. This disease has not yet been described in pinnipeds however seems very possible in this case.

16/07/2018:

A fluorescein test was performed and showed a positive result.

26/07/2018:

Start stem cell therapy. In the first week during this period an error occurred. This resulted in a seal receiving a whole vial of approximately 2-3 ml due to the cap of the vial that came loose during application.

09/08/2018:

Stem cell therapy was stopped.

3 Information from https://www.rashondenwijzer.nl/aandoeningen/plasmacellulaire-conjunctivitis- ontsteking-derde-ooglid on 21 March 2019.

25/08/2018:

A purulent conjunctivitis in which fibrin partially overgrown the cornea occurred. Therapy was initiated with antibiotic (amoxycillin) and pain relief (meloxicam + tramadol).

11/09/2018:

The sclera became more hypertrophic. The cornea was visually reduced and necrosis of the cornea could be seen at this stage. New tissue seemed to overgrow the cornea. A possible overstimulation of the fibroblasts due to an overload of stem cells arises as a potential hypothesis.

08/02/2019:

The purulent conjunctivitis was successfully treated with the antibiotics however all other eye lesions are still present in this seal.

Discussion To start, the eyes were never checked by an experienced ophthalmologist. Diagnoses were made on what was macroscopically visible. A dark room, a focal light source, a direct ophthalmoscope and lens were not present. It might have been interesting to check the eyes at night-time as a solution for the absent dark room. Both seals and both eyes have complex problems; They have glaucoma, lens luxation, extensive keratitis and ulcerations. The ulcerations are very likely secondary to old lesions that were not treated (correctly) in the past. It is difficult to form a correct approach on how to treat these animals sufficiently. Looking back on it, these animals were not the best choice for this study. The purpose of the stem cells was to heal the ulcerations, however this gave little to no result since there is a worse underlying problem that cannot be solved easily. Furthermore, lensectomy should have been performed in the past to improve vision, relieve pain and prevent secondary ocular conditions. The dose of stem cells administered in both seals was in agreement with the producer of the eye drops. The duration of the administration was decided by the veterinarian of the park. Since this is the first recorded application of stem cell therapy in pinnipeds the dose and duration might not have been optimal. Further research with stem cell therapy should be done in more adequate pinnipeds, thus suffering from only an ulceration, and be monitored with fluorescein tests to follow up the possible healing effect. Both animals have both sided severe eye lesions, it is unclear why full enucleation of the eyes was not performed (yet). The visibility of these animals is suspected to be very low to non-existent. Seals with one eye are still able to hunt in the wild and completely blind seals in captivity can adapt very quickly using tactile and acoustic cues. Enucleation is a procedure relatively often done in pinnipeds. Since both seals suffer from severe and painful lesions, enucleation would be very beneficial. Possible reasons why this has not been done could be because of an economic perspective and fear of misperception by the visitors in the park. The operation of both animals would cost money and second to that it might have an impact on the perception of the visitors of the park who could think about an eyeless seal as unpleasant. Less visitors mean less profit so this could then also be traced down to an economic aspect.

Conclusion Eye problems in pinnipeds in captivity are a very prominent and important issue. Cases like these are no exceptions. A correct diagnosis should be made based on more objective techniques. The stem cell therapy wasn’t a success in these two animals. In future research it would be interesting to follow a case with (a) pinniped(s) that is first correctly diagnosed and secondly only suffers from an ulceration. I believe stem cells are promising in treatment of eye ulcerations however further research is needed.

Appendix I

Dear,

My name is Delphine Boone and I’m a veterinary student at Ghent University. For my master thesis about eye injuries of Pinnipedia I’m trying to collect as much data as possible about this subject with use of a short survey. This thesis is under supervision of Dr. Piet De Laender who, among other things, works as a veterinarian at “Sea Life” in Blankenberge and “Boudewijn Seapark” in Bruges. Your help would be extremely appreciated and I can promise you a mention –unless not wanted- in my thesis.

1) How many pinnipeds are there at the facility?

2) If there are animals with eye injuries, in which category do they belong and what is the exact number of animals per category? Does this concern male or female animals?

Degenerative?

Traumatic?

Infectious?

And more specific:

Glaucoma?

Corneal oedema?

Corneal ulceration?

Cataract?

Panophthalmitis?

Lens luxation?

Extra: pictures or some information about the possible eye injuries are also very welcome.

Thank you very much!

Delphine Boone

Cher,

Je me présente: je suis Delphine Boone et pour le moment je suis étudiante vétérinaire à l’Université de Gand. Dans le cadre de ma dissertation à propos des lésions oculaires chez les phoques et les otaries, j’essaise d’avoir le plus possible de données en utilisant cette courte enquête. Cette dissertation est entre autres sous la direction de vétérinaire Piet De Laender, qui est lié à Sea Life à Blankenberge et Boudewijn Seapark à Bruges. Votre aide serait fortement apprécié et une mention sera faite dans la dissertation, sauf si vous ne le souhaitez pas.

1) Combien de pinnipèds il y a sur place?

2) S’il y a des animaux avec des lésions oculaires, sous quelle catégorie tombent-ils? Et combien d’animaux y a t’il sous cette catégorie? Mâles ou femelles?

Dégénération?

Traumatismes?

Infectieux?

Et plus spécifique:

Glaucome?

Oedème cornéen?

Ulcération de la cornéen?

Cataracte?

Panophtalmie?

Luxation du cristallin?

Si vous avez des images ou plus d’information sur les lesions oculaires, ceci sera très apprécié.

Je vous remercie d’avance pour votre cooperation.

Veuillez agréer, messieurs, mesdames, mes sincères salutations.

Delphine Boone

Beste,

Mijn naam is Delphine Boone en ik ben een studente diergeneeskunde aan de universiteit van Gent. In het kader van mijn masterproef omtrent oogletsels bij zeehonden en zeeleeuwen tracht ik a.d.h.v. een korte enquête zoveel mogelijk gegevens te verzamelen m.b.t. dit onderwerp. Deze masterproef is o.a. onder begeleiding van Dr. Piet De Laender, verbonden met zowel “Sea Life” te Blankenberge als “Boudewijn Seapark” te Brugge. Uw hulp zou enorm geapprecieerd worden en een vermelding - tenzij u deze niet wenst - in mijn masterproef kan ik u beloven.

1) Hoeveel Pinnipedia zijn er momenteel aanwezig in het park?

2) Indien er dieren aanwezig zijn met oogletsels onder welke categorie vallen deze en wat is het exacte aantal dieren bij desbetreffende categorie? Gaat het om mannelijke of vrouwelijke dieren?

Degeneratief?

Traumatisch?

Infectieus?

En meer specifiek:

Glaucoom?

Cornea oedeem?

Cornea ulceratie?

Cataract?

Panoftalmie?

Lensluxatie?

Extra: fotomateriaal of informatie over de eventuele oogletsels is zeker welkom.

Hartelijk bedankt!

Delphine Boone

Appendix II

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