They are grateful to Bruce Brown and Rachel Brown of the Environment Agency for carrying out pollutant analy- ses, and the staff at VLA â Shrewsbury for ...
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Retinal dysplasia in wild otters (Lutra lutra) D. L. Williams, V. R. Simpson, A. Flindall Eyes from 88 otters found dead in south-west England between 1990 and 2000, were collected as part of a larger pathological study. Histopathological examination of 131 eyes revealed dysplastic changes such as rosetting and folding in the retinas of 26 of the otters. In the eyes of 42 of the otters there were postmortem and fixation-induced retinal detachment which complicated the differentiation of dysplastic from normal retina, but 11 eyes had folds which probably indicated a dysplastic pathology. The eyes of 18 of the otters had inflammatory or autolytic changes which precluded a definitive evaluation of their dysplastic status. Liver samples from 55 of the otters were analysed for a range of polychlorinated hydrocarbons and for vitamin A. The otters with dysplastic retinas had significantly lower concentrations of vitamin A and higher concentrations of dieldrin than the otters with normal retinas.
IT is widely believed that the pan-European decline in wild otters from the late 1950s was caused by the agricultural use of organochlorine pesticides and the industrial use of polyclorinated biphenyls (PCBs), although no definitive proof has been reported of pathological effects caused in otters by either of these groups of chemicals. However, there is good evidence that they have a deleterious effect on vitamin A metabolism (Kimborough 1974, Hëkansson and others 1992). No pathological effects have been reported in otters which could be associated either with direct toxicity due to halogenated hydrocarbons or with indirect effects due to changes caused by reduced levels of vitamin A. This paper describes dysplastic retinal lesions in wild otters which are correlated statistically with low levels of vitamin A and high levels of dieldrin. MATERIALS AND METHODS The eyes were collected from 88 wild otters found dead in south-west England between 1990 and 2000. The gross pathological findings in many of these cases have been described by Simpson (1997). Between February 1990 and May 1997, one eye was collected from 45 otters (group 1) and fixed in 10 per cent buffered formol saline. From May 1997, both eyes were collected from 43 otters (group 2); one was fixed in 10 per cent buffered formol saline and transferred to 70 per cent 52
alcohol for 24 hours before it was bisected, the other was fixed in 2·5 per cent gluteraldehyde in phosphate-buffered saline. They were examined before being bisected and the diameter of the globe was measured with calipers. The globes were processed routinely and embedded in paraffin wax; 8 µm sections were cut and stained with haematoxylin and eosin by standard histological techniques. One section from each eye was evaluated for the presence of retinal dysplasia; in five of the eyes in which a diagnosis of retinal dysplasia was made, five sections were taken at intervals of 500 µm to assess the degree to which the histological appearance in one section was representative of that in the entire eye and to determine whether the retinal lesions were rosettes or artefactual folds. The eyes of 20 badgers found as road casualties were examined as a control population to determine the postmortem changes in animals which had died a significant time before their tissues were fixed. Samples of liver were collected at the postmortem examination of 55 of the otters and frozen before the concentrations of vitamin A levels and pollutants (dieldrin, pp’-DDE, pp’-DDT, pp’-TDE, gamma HCH, PCB congeners 105, 118, 138, 153, 156 and 180) were determined as described by Simpson and others (2000). The vitamin A levels in otters with normal eyes and otters with eyes with dysplastic retinal lesions were compared by using a two-tailed t test on log-square normalised data (Petrie and Watson 1999). The Veterinary Record, July 10, 2004
Veterinary Record (2004) 155, 52-56 D. L. Williams, MA, VetMB, PhD, CertVOphthal, MRCVS, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES V. R. Simpson, BVSc, CBiol, FIBiol, DTVM, MRCVS, Jollys Bottom Farm, Station Road, Truro TR4 8PB
A. Flindall, Centre for Preventative Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU
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(a)
(c)
RESULTS Gross pathological findings Few of the eyes had significant abnormalities on gross examination of the whole or half globe; five had gross inflammatory disruption of the posterior segment and in 18 the retina was clearly detached; 16 had grossly visible lens opacities. The mean (sd) diameter of the globes was 10 (1·4) mm. There were no apparent correlations between the gross intraocular pathology or measurements of globe diameter and the concentrations of vitamin A or pesticides or PCBs in liver. Histopathological findings Retinal folds and rosettes characteristic of retinal dysplasia were observed in the eyes of 26 of the 88 otters. However, in a substantial number of eyes, the abnormalities in retinal development were complicated by other intraocular pathol-
FIG 2: Retinal pigment epithelial hypertrophy characteristic of antemortem retinal detachment, rather than a postmortem or fixation artefact. Haematoxylin and eosin. x 1000
The Veterinary Record, July 10, 2004
(b)
FIG 1: (a) Two adjacent single-layer rosettes, x 300, (b) one double-layer retinal rosette, x 300, (c) one double-layer retinal rosette, x 500, each characteristic of retinal dysplasia. Haematoxylin and eosin
ogy or artefactual postmortem or fixation-induced changes. Four categories of histopathological change were distinguished. First, eyes in which retinal rosettes were obvious (Fig 1) were considered to have retinal dysplasia. Secondly, eyes with rosettes but also with detachment and folding were also considered to show retinal dysplasia. The presence of retinal pigment epithelial hypertrophy (Fig 2) in eyes from these two groups strongly suggested that retinal detachment was premortem, rather than a postmortem artefact. A third group consisted of eyes with retinal detachment alone, and without retinal pigment hypertrophy (Fig 3). These eyes were considered to be showing postmortem or fixation-induced artefactual changes. Most eyes in all groups showed vacuolated change in the retinal cellular layers (Fig 1) which was considered to be early autolytic change. A fourth group was eyes with autolysis or inflammatory disruption. The number of eyes with these changes is given in Table 1. None of the badger eyes showed changes indicative of retinal dysplasia; the only changes observed were either postmortem changes or fixation effects. In the five otter eyes from which multiple sections were taken, 18 of the 25 sections (72 per cent) had lesions indicative of retinal dysplasia, giving a false negative rate of 28 per cent. Eleven per cent of the eyes had other non-dysplastic pathology of the ocular surface or anterior and posterior segments. Such lesions included lymphoid aggregates in the cornea of the eyes from two otters, lenticular vacuoles and posterior capsular aggregations in five eyes, neurectodermal proliferation and choroidal thickening in one eye, multinucleate giant cell granulomas in the extraocular musculature of one eye and protozoal cysts in the extraocular muscle of one eye. Correlation with vitamin A and pollutant levels The mean (sd) concentration of vitamin A in the livers of the otters with dysplastic retinas was 81 (104) µmol/kg compared with 465 (482) µmol/kg in the normal otters; the difference was significant at P=0·0011 (Fig 4). There was no significant difference between the total concentrations of PCB congeners in the livers of the otters with dysplastic and normal retinas. However, the mean hepatic concentration of dieldrin in the otters with dysplastic retinas was 339 (305) µg/kg, over three times the concentration in the otters with normal eyes (90·1 [67·4] µg/kg) (P=0·028). This finding is to be expected given the inverse correlation between the hepatic concentrations of dieldrin and vitamin A (Simpson and others 2000) (Fig 5). 53
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DISCUSSION The rapid decline in otter populations across Europe in the 1950s and 1960s is thought to have been causally linked to the use of industrial PCBs and/or organochlorine pesticides, particularly the chemicals dieldrin and aldrin, which is metabolised to dieldrin. In Britain the most rapid decline occurred after the introduction of dieldrin in 1956 (Chanin and Jefferies 1978) and the population has recovered since the partial ban on the use of these pesticides in 1981 and the complete ban in 1989 (Strachan and Jefferies 1996). Other halogenated hydrocarbons, and in particular certain PCB congeners, are undoubtedly important, but healthy otter populations occur in areas such as the Shetlands where PCB levels are high but dieldrin contamination is low (Kruuk and Conroy 1996). Organochlorine pesticides and PCBs have a toxic effect through their influence on vitamin A metabolism. Studies on the toxic effects of dieldrin in the pigeon (Columba livia) showed that there was an initial increase in hepatic vitamin A followed by a decrease after chronic exposure (Jefferies 1975); PCB congeners also reduce hepatic vitamin A storage and increase its renal excretion by blocking binding sites on a transport protein in plasma (Brouwer and others 1986). There is evidence of an inverse correlation between vitamin A levels and PCBs in otters from Denmark (Murk and others 1998) and between PCBs and organochloride pesticides and vitamin A in otters in south-west England (Simpson and others 2000). Thus, the higher concentration of dieldrin in the otters with dysplastic retinas in this report is not surprising. The pathology of chronic vitamin A deficiency includes reproductive disorders such as fetal resorption, abortion and stillbirth, abnormal bone remodelling, hydrocephalus, gonadal hypoplasia and cryptorchidism. Ocular lesions include xerophthalmia, keratitis and retinal degeneration (Hayes 1974). When the developing fetus or young animals become vitamin A deficient, abnormal development can result in retinal dysplasia, as has been reported in piglets and calves on deficient diets (Palludan 1961, Van der Lugt and Prozetsky 1989), and cranial bone abnormalities with a narrowing of the skull foraminae have been reported in calves (Spratling and others 1965, Barnett and others 1970) and in puppies (Mellanby 1941) and piglets (Palludan 1961). Retinal dysplasia literally means maldevelopment of the retina, but the term has been used to denote changes involving retinal rosettes, folds and gliosis since it was first described at the end of the 19th century. In human ophthalmology it has been used to cover retinal maldevelopment with several aetiologies, including defects associated with chromosomal abnormalities caused by the teratogenic effects of drugs (Percy and Danylchuk 1977, Chan and others 1978) and irradiation (Goldstein and Wexler 1931, Silverstein and others 1971a, Lahav and others 1973, Shimada and others 1973, Gorthy 1979). In the veterinary literature, retinal dysplasia has been used to describe similar retinal changes inherited in a number of dog breeds, sometimes as multifocal vermiform lesions of the fundus (Ashton and others 1968, Heywood and Wells 1970), sometimes as larger areas of retinal detachment (Bedford 1982, O’Toole and others 1983, Long and Crispin 1999), sometimes as complete retinal detachment (Ashton and others 1968, Rubin 1968, Aroch and others 1996), and sometimes as retinal lesions associated with systemic syndromes in the labrador retriever and samoyed breeds (Carrig and others 1977, Meyers and others 1983, Du and others 2000) or nonsyndromic multiocular anomalies (Peiffer and Fischer 1983, Bergsjo and others 1984, Laratta and others 1985). Infections with herpesvirus in dogs (Percy 1971) and bluetongue virus in sheep (Silverstein and others 1971b) can give rise to lesions termed retinal dysplasia but which are, in effect, malformations due to virally induced retinal necrosis. The ocular teratogenic influence of vitamin A deficiency has been reported to cause 54
FIG 3: Postmortem retinal detachment without retinal dysplasia, presumed to be induced by the process of fixation. Haematoxylin and eosin. x 100
signs of retinal dysplasia in several species and more severe malformations from microphthalmia to anophthalmia (Palludan 1961, Van der Lugt and Prozetsky 1989). Any microphthalmic eye with multiple congenital anomalies may be characterised by dysplastic areas of retina (Peiffer and Fischer 1983, Kaswan and others 1987, Buyukmihci and others 1988, Weiss and others 1989, Williams and Barnett 1993). Given that retinal folds are a focal abnormality it might be argued that examining one section from each eye would give an erroneous estimation of the occurrence of retinal abnormalities. In the five eyes from each of which five sections were taken, a false negative result was obtained in seven sections (28 per cent). This apparently low sensitivity suggests that the percentage of affected animals in this report may be an underestimate. A second factor which complicated the diagnosis of retinal dysplasia in several of the otters was the artefactual retinal folding and detachment which occurred as a result of postmortem changes and artefacts induced by fixation. The interval between death and fixation in these road casualties accounts for the postmortem autolysis and detachment. Trauma may have been a factor in the retinal disruption and inflammatory changes observed in some eyes. Fixation-induced changes could readily account for the retinal detachment observed in 38 per cent of the eyes. Szczech and others (1976) reported artefactual retinal folding in rat fetuses produced by fixation in 70 per cent alcohol. Fixation in 4 per cent buffered formaldehyde can cause retinal detachment in adult eyes through osmotic effects (Margo and Lee 1995), but the immersion in 70 per cent alcohol used in this study for post-fixation scleral hardening, is unlikely to have caused retinal artefacts. However, the formalin used as an initial fixative may have caused some of the total retinal detachments observed, because there was a higher proportion of retinal detachments in the formalin-fixed eyes (41 per cent) than in the gluteraldehyde-fixed eyes (11·5 per cent). Gross retinal folding and total retinal detachment may thus be a result of osmotic changes during fixation. The rosettes observed could have been the result of the section passing
TABLE 1: Numbers (percentage) of otter and badger eyes with different categories of lesion Group 2 Group 2 All otter Group 1 (formalin) (gluteraldehyde) eyes
Category of lesion Number of animals No abnormality detected Retinal dysplasia alone Retinal dysplasia with folds and detachment Retinal pigment epithelial hypertrophy Postmortem detachment alone Inflammatory or autolytic disruption
All otters
Badger eyes
45 6 (13) 9 (20)
43 6 (14) 8 (19)
43 20 (47) 9 (21)
131 88 24 32 (24·5) 10 (11·5) 7 (29) 26 (20) 18 (20·5) 0
4 (9)
3 (7)
4 (9)
11 (8)
8 (9)
0
4 (9)
1 (2)
4 (9)
9 (7)
7 (8)
0
16 (35)
18 (41)
5 (11·5)
39 (30)
10 (23)
8 (19)
5 (11·5)
23 (17·5) 18 (20·5)
The Veterinary Record, July 10, 2004
34 (38·5) 16 (66) 1 (5)
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4
Log vitamin A concentration (µmol/kg) Log vitamin A concentration (µmol/kg)
4
Concentration of vitamin A (log Concentration of vitamin A (log 10 µmol/kg) 10 µmol/kg)
3
2
1
3
2
1
0
–1 1
2 3 Log Logdieldrin dieldrinconcentration concentration(µmol/kg) (µg/kg)
4
FIG 5: Relationship between the concentrations of vitamin A and dieldrin in the livers of the otters 0
FIG 4: Log10 concentrations of vitamin A in the liver of otters with normal and dysplastic retinas
–1 Dysplastic Dysplastic NormalNormal Retina Retina
through folded retina. The serial sections taken to assess the validity of this finding show, however, that the lesions are evidence of retinal rosettes and not large folds cut several times. The fact that these rosettes were not observed in the badgers, for which there was a similar delay between death and fixation and the same fixation procedure was used, strongly suggests that the rosettes were lesions and not postmortem or fixation and processing artefacts. Similar questions might be raised about the low levels of vitamin A in the livers of animals analysed some time after death. However, the livers were all in a fresh state on gross examination and did not show substantial postmortem signs of autolysis on histological examination. There is evidence that the levels of vitamin A in liver samples taken postmortem do not decrease significantly with time (Underwood and others 1970, Raica and others 1972) and thus the levels reported are likely to indicate the levels in the living animals. Having dismissed artefactual changes as the cause of the dysplastic changes observed, the retinal lesions could potentially have been either inherited or induced by infectious, nutritional or toxic factors. The first possibility is unlikely in outbred wild animals, although, given the fragmented otter population, it cannot be completely excluded. Viral infections have been reported to cause retinal dysplasia, but the lesions produced are more those of retinal disorganisation than the well-defined rosettes observed. The correlation of the retinal pathology with low hepatic levels of vitamin A strongly suggests that hypovitaminosis A may have been a contributory factor in the genesis of the retinal lesions; the affected otters had a substantially lower mean level of hepatic vitamin A, but the mechanism of its effect is unclear. Retinal dysplasia develops during the late gestational and early neonatal period, with low maternal levels of vitamin A resulting in low intrauterine fetal exposure to vitamin A and low vitamin A levels in the milk ingested by the young otter pups. The hepatic vitamin A levels in adult otters with retinal dysplasia may not be as relevant as the levels when in utero or as suckling cubs, data which are not available. The Veterinary Record, July 10, 2004
The hypovitaminosis A is probably associated with the toxicity of halogenated hydrocarbons, because there is considerable evidence linking abnormal vitamin A metabolism with PCBs and organochlorine toxicity. The high levels of these chemicals in colostrum may have a major effect on the developing cub, and particularly affect the development of the retina much of which occurs in the early postnatal period. However, it is difficult to correlate low levels of vitamin A or their associated retinal pathology with the total burden of PCBs and organochlorine pesticides because different PCB congeners and isomers of organochlorine pesticides affect vitamin A metabolism to different degrees. It is therefore difficult to assess the effects of a mixture of congeners and it is necessary to calculate a toxic equivalence quotient or TEQ, which requires the measurement of the concentrations of a number of PCB congeners. This has not been possible for most of the otters in the present study. However, the organochlorine pesticides and in particular dieldrin have been shown to have an inverse correlation with levels of vitamin A (Simpson and others 2000), and a correlation between retinal pathology and dieldrin levels would therefore suggest that the retinal pathology is associated not only with low vitamin A levels but also with PCB and/or organochlorine toxicity. This was the case, with the otters with dysplastic retinas having approximately three times as high a concentration of dieldrin in their livers as the otters with normal retinas (P=0·028). The corneal inflammatory changes observed could be attributed to trauma, and the cysts in the periorbital musculature could be attributed to parasites. It was difficult to determine whether the lenticular lesions observed postmortem and histopathologically were inherited, nutritional or artefactual lesions, and it is possible that they were also related to hypovitaminosis A during development. The finding of developmental retinal abnormalities in these otters which were correlated with low levels of vitamin A and high levels of dieldrin in their livers, provides the first documented evidence that halogenated hydrocarbons have a toxic effect on otters through their effects on vitamin A levels. ACKNOWLEDGEMENTS The authors wish to thank the many people who assisted in the collection of otter carcases, including staff of the county Wildlife Trust, the RSPCA and, in particular, the Environment 55
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Agency. They are grateful to Bruce Brown and Rachel Brown of the Environment Agency for carrying out pollutant analyses, and the staff at VLA – Shrewsbury for vitamin A analyses and at VLA – Truro for laboratory support. They also wish to thank the Environment Agency for funding this work. References AROCH, I., OFRI, R. & AIZENBERG, I. (1996) Haematological, ocular and skeletal abnormalities in a Samoyed family. Journal of Small Animal Practice 37, 333-339 ASHTON, N., BARNETT, K. C. & SACHS, D. D. (1968) Retinal dysplasia in the Sealyham terrier. Journal of Pathology and Bacteriology 96, 269-272 BARNETT, K. C., PALMER, A. C., ABRAMS, J. T., BRIDGE, P. S., SPRATLING, F. R. & SHARMAN, I. M. (1970) Ocular changes associated with hypovitaminosis A in cattle. British Veterinary Journal 126, 561-573 BEDFORD, P. G. C. (1982) Multifocal retinal dysplasia in the rottweiler Veterinary Record 111, 304-305 BERGSJO, T., ARNESEN, K., HEIM, P. & NES, N. (1984) Congenital blindness with ocular developmental anomalies, including retinal dysplasia, in Doberman Pinscher dogs. Journal of the American Veterinary Medical Association 184, 1383-1386 BROUWER, A., VAN DEN BERG, K., BLANER, W. & GOODMAN, D. (1986) Transthyretin (prealbumin) binding of PCBs. A model for the mechanism of interference with vitamin A and thyroid hormone metabolism. Chemosphere 15, 1699-1706 BUYUKMIHCI, N. C., MURPHY, C. J. & SCHULZ, T. (1988) Developmental ocular disease of raptors. Journal of Wildlife Diseases 24, 207-213 CARRIG, C. B., MACMILLAN, A., BRUNDAGE, S., POOL, R. R. & MORGAN, J. P. (1977) Retinal dysplasia associated with skeletal abnormalities in Labrador retrievers. Journal of the American Veterinary Medical Association 1570, 49-57 CHAN, C. C., FISHMAN, M. & EGHBERT, P. R. (1978) Multiple ocular anomalies associated with maternal LSD ingestion. Archives of Ophthalmology 96, 282-284 CHANIN, P. & JEFFERIES, D. (1978) The decline of the otter Lutra lutra L in Britain: an analysis of hunting records and discussion of causes. Biological Journal of the Linnean Society 10, 305-328 DU. F., ACLAND, G. M. & RAY, J. (2000) Cloning and expression of type II collagen mRNA: evaluation as a candidate for canine oculo-skeletal dysplasia. Gene 255, 307-316 GOLDSTEIN, I. & WEXLER, D. (1931) Rosette formation in eyes of irradiated human embryos. Archives of Ophthalmology 5, 591-600 GORTHY, W. C. (1979) Developmental ocular abnormalities in rats with X rayinduced cataract mutation. Investigative Ophthalmology and Visual Science 18, 939-946 HAYES, K. C. (1974) Retinal degenerations in monkeys induced by deficiencies in vitamins A and E. Investigative Ophthalmology and Visual Science 13, 499-510 HËKANSSON, H., MANZOOR, E. & AHLBORG, U. G. (1992) Effects of technical PCB preparations and fractions thereof on vitamin A levels in the mink (Mustela vison) Ambio 21, 588-590 HEYWOOD, R. & WELLS, G. A. (1970) A retinal dysplasia in the Beagle dog. Veterinary Record 87, 178-180 JEFFERIES, D. (1975) The role of the thyroid. In Organochlorine Insecticides: Persistent Organic Pollutants. Ed F. Moriarty. London, Academic Press. pp 175-182 KASWAN, R. L., COLLINS, J. G., BLUE, J. L. & MARTIN, C. L. (1987) Multiple hereditary ocular anomalies in a herd of cattle. Journal of the American Veterinary Medical Association 191, 97-99 KIMBOROUGH, R. (1974) The toxicity of polychlorinated polycyclic compounds and related chemicals. CRC Critical Reviews 2, 445-495 KRUUK, H. & CONROY, J. (1996) Concentrations of some organochlorines in otters (Lutra lutra) in Scotland: implications for populations. Environmental Pollution 92, 165-171 LAHAV, M., ALBERT, D. M. & WYLAND, S. (1973) Clinical and histopathological classification of retinal dysplasia. American Journal of Ophthalmology 75, 648-667 LARATTA, L. J., RIIS, R. C., KERN, T. J. & KOCH, S. A. (1985) Multiple congenital ocular defects in the Akita dog. Cornell Veterinarian 75, 381-392 LONG, S. E. & CRISPIN, S. M. (1999) Inheritance of multifocal retinal dysplasia in the golden retriever in the UK. Veterinary Record 145, 702704 MARGO, C. E. & LEE, A. (1995) Fixation of whole eyes: the role of fixation osmolarity in the production of tissue artefacts. Graefes Archives of Clinical and Experimental Ophthalmology 233, 366-370 MELLANBY, E. (1941) Skeletal changes affecting the nervous system produced
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in young dogs by diets deficient in vitamin A. Journal of Physiology 99, 467486 MEYERS, V. N., JEZYK, P. F., AGUIRRE, G. D. & PATTERSON, D. F. (1983) Short-limbed dwarfism and ocular defects in the Samoyed dog. Journal of the American Veterinary Medical Association 183, 975-979 MURK, A. J., LEONARDS, P. E. G., VAN HATTUM, B., LUIT, R., VAN DER WEIDEN, M. E. J. & SMIT, M. (1998) Application of biomarkers for exposure and effect of polyhalogenated aromatic hydrocarbons in naturally exposed European otters (Lutra lutra). Environmental Toxicology and Pharmacology 6, 91-102 O’TOOLE, D. O., YOUNG, S., SEVERIN, G. A. & NEUMANN, S. (1983) Retinal dysplasia of English springer spaniel dogs: light microscopy of the postnatal lesions. Veterinary Pathology 20, 298-311 PALLUDAN, B. (1961) The teratogenic effect of hypovitaminosis A in pigs. Acta Veterinaria Scandanavica 2, 32-59 PEIFFER, R. L., Jr & FISCHER, C. A. (1983) Microphthalmia, retinal dysplasia, and anterior segment dysgenesis in a litter of Doberman Pinschers. Journal of the American Veterinary Medical Association 183, 875-878 PERCY, D. H. (1971) Lesions in puppies surviving infections with canine herpes virus. Veterinary Pathology 8, 37-53 PERCY, D. H. & DANYLCHUK, K. D. (1977) Experimental retinal dysplasia due to cytosine arabinoside. Investigative Ophthalmology and Visual Science 16, 353-363 PETRIE, A. & WATSON, P. (1999) Statistics for Veterinary and Animal Science. Oxford, Blackwell Science. p 85 RAICA, N., Jr, SCOTT, J., LOWRY, L. & SAUBERLICH, H. E. (1972) Vitamin A concentration in human tissues collected from five areas in the United States. American Journal of Clinical Nutrition 25, 291-296 RUBIN, L. F. (1968) Heredity of retinal dysplasia in Bedlington terriers. Journal of the American Veterinary Medical Association 152, 260-262 SHIMADA, M., WAKAIZUMUI, S., KASUBUCHI, Y., KUSUNOKI, T. & NAKAMURA, T. (1973) Cytosine arabinoside and rosette formation in mouse retina. Nature 246, 151-152 SILVERSTEIN, A. M., OSBURN, B. I. & PRENDERGAST, R. A. (1971a) The pathogenesis of retinal dysplasia. American Journal of Ophthalmology 72, 1319 SILVERSTEIN, A. M., PARSHALL, C. J., OSBURN, B. I. & PRENDERGAST, R. A. (1971b) An experimental virus-induced retinal dysplasia in the fetal lamb. American Journal of Ophthalmology 72, 22-34 SIMPSON, V. R. (1997) Health status of otters (Lutra lutra) in south-west England based on postmortem findings. Veterinary Record 141, 191-197 SIMPSON, V. R., BAIN, M. S., BROWN, R., BROWN, B. F. & LACEY, R. F. (2000) A long term study of vitamin A and polychlorinated hydrocarbon levels in otters (Lutra lutra) in south west England. Environmental Pollution 110, 267-275 SPRATLING, F. R., BRIDGE, P. S., BARNETT, K. C., ABRAMS, J. T., PALMER, A. C. & SHARMAN, I. M. (1965) Experimental hypovitaminosis A in calves. Veterinary Record 77, 1532-1542 STRACHAN, R. & JEFFERIES, D. J. (1996) Otter Survey of England 1991-1994. London, Vincent Wildlife Trust. p 223 SZCZECH, G. M., PURMALIS, B. P. & CARLSON, R. G. (1976) Folded retinas in rat fetuses: artefacts produced by fixation in alcohol. Toxicological and Applied Pharmacology 35, 347-354 UNDERWOOD, B. A., SIEGEL, H., DOLINSKI, M. & WEISELL, R. C. (1970) Liver stores of alpha-tocopherol in a normal population dying suddenly and rapidly from unnatural cases in New York City. American Journal of Clinical Nutrition 23, 1314-1321 VAN DER LUGT, J. J. & PROZETSKY, L. (1989) The pathology of blindness in new-born calves caused by hypovitaminosis A. Ondesterpoort Journal of Veterinary Research 56, 99-109 WEISS, A. H., KOUSSEFF, B. G., ROSS, E. A. & LONGBOTTOM, J. (1989) Complex microphthalmos. Archives of Ophthalmology 107, 1619-1624 WILLIAMS, D. L. & BARNETT, K. C. (1993) Bilateral optic disc colobomas and microphthalmos in a thoroughbred horse. Veterinary Record 132, 101-103
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