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Characterization and causal investigations of an alopecia syndrome in Australian fur seals (Arctocephalus pusillus doriferus)

Michael Lynch , Roger Kirkwood , Rachael Gray , David Robson , Greg Burton , Leslie Jones , Rodney Sinclair , John P. Y. Arnould
DOI: http://dx.doi.org/10.1644/11-MAMM-A-279.1 504-513 First published online: 30 April 2012

Abstract

Fur seals rely on pelage consisting of dense, fine underfur protected by guard hairs as their primary means of limiting thermoregulatory cost. A distinctive syndrome of alopecia occurs at high prevalence in 1 colony of Australian fur seals (Arctocephalus pusillus doriferus). It is characterized by bilaterally symmetrical hair loss on the dorsal body surface and a biased prevalence toward juvenile females. Light and scanning electron microscopy demonstrated that alopecia is due to fracture of the hair shaft above the skin level. No evidence of viral, bacterial, fungal, or parasite infection was found and histological examination of skin biopsies revealed no pathological variation between case and control seals. Affected animals had statistically significant lower tyrosine and zinc concentrations in hair than unaffected seals. This may increase hair brittleness and, therefore, predispose its fracture. Alopecia cases also had higher levels of heavy metals and persistent organic pollutants, which may indicate they forage in ecosystems where concentrations of pollutants are higher.

Key words
  • alopecia
  • Australian fur seals
  • disease
  • hair
  • nutrition
  • pinniped
  • toxin
  • trace element

Marine mammals control heat loss in the aquatic environment through numerous anatomical and physiological adaptations of the integument and circulatory system (Pabst et al. 1999). Fur seals rely on pelage consisting of dense, fine underfur protected by thicker, flattened, guard hairs as their primary means of limiting thermoregulatory cost (Ling 1970). The fact that all fur seal species molt annually, itself an energetically demanding process, is evidence of the biological importance of an intact and functional pelage (Boily 1996; Ling 1970). Damage or loss of the pelage places additional energy demands upon the individual. Diversion of a greater proportion of energy to thermoregulation may compromise foraging ability and increase catabolism of body tissues, increasing mortality risk (Rosen et al. 2007).

A distinctive syndrome of bilaterally symmetrical alopecia (hair loss) occurs in Australian fur seals (Arctocephalus pusillus doriferus) on Lady Julia Percy Island in northwestern Bass Strait (Lynch et al. 2011). Lady Julia Percy Island is 1 of the 2 largest Australian fur seal breeding colonies, with a population in the order of 30,000 seals, equating to approximately 25% of the total population for this species (Kirkwood et al. 2010). The alopecia syndrome primarily affects juvenile females but is also observed at a lower prevalence in adult females. It is not seen in postpubescent males and only occasionally in juvenile males. Affected animals show guard hair loss over the dorsal thorax (Fig. 1) and, in severe cases, this extends over most of the back and head. Up to 50% of juvenile females at Lady Julia Percy Island are affected, compared to ≤3% prevalence at other Australian fur seal colonies. Alopecie seals do not adequately limit heat loss from affected areas and are in poorer body condition than nonaffected animals. An alopecia syndrome such as this described in the Australian fur seal had not previously been reported in a pinniped species and, may have the potential to act as a population regulatory factor at this colony.

Fig. 1.

Dorsal surface of an Australian fur seal with alopecia. Guard hairs have been lost in a patch on the thoracic area and the underfur is exposed.

Infection with viral, bacterial, or parasitic pathogens that cause focal or extensive dermatitis can cause alopecia in pinnipeds (Dailey 2001; Dunn et al. 2001; Simpson et al. 1994). Hair is directly shed from inflamed follicles or skin irritation may encourage self-trauma resulting in hair fracture. Also, fungal infection can cause patchy alopecia in captive pinnipeds (Guillot et al. 1998; Montali et al. 1981). Fungal hyphae colonize the hair shaft, weakening its structure with subsequent fracture. Diagnosis of infectious causes of alopecia is usually made by isolation of the pathogen or parasite, in addition to demonstration of characteristic pathology in histological skin sections.

Noninfectious causes of alopecia include endocrine disturbances, nutritional deficits or toxicities, and immune-mediated disease. Bilaterally symmetrical alopecia is a common clinical symptom of sex hormone or thyroid hormone disturbances that sporadically occur in humans and domestic animals, some of which have genetic components that influence their expression (Frank et al. 2003; Greco 2007; Shapiro 2007). Descriptions of endocrine disruption in marine mammals generally concern the effect of environmental toxins on thyroid hormone homeostasis (Hall et al. 2003; Routti et al. 2010). Links between endocrine-disruptive toxins and alopecia, however, have not been established in pinnipeds. Most nutritionally related causes of alopecia result from deficiencies of amino acids, trace elements, vitamins, or fatty acids that are essential to the normal growth and structure of skin and hair (Goldberg and Lenzy 2010; Puls 1994). It is well recognized that pinnipeds undergo temporal fluctuations in nutrient availability and that long-term changes in prey composition may negatively impact the health of some populations (Trites and Donnelly 2003). In addition, the absorption and bioavailability of some nutrients can be affected by ingested toxins (Debier et al. 2005). Deficiencies or toxicities of specific nutrients have not been linked with dermatological syndromes in free-ranging pinnipeds, although experimentally induced vitamin E deficiency in captive harp seals (Pagophilus groenlandicus) resulted in irregular and incomplete molt cycles (Engelhardt and Geraci 1978). Immune-mediated skin disease is a common cause of alopecia in domestic mammals with dermatological problems (Scott and Paradis 1990). Affected individuals are often pruritic and alopecia is typically due to hair fracture resulting from self-trauma. Immune-mediated skin disease has been suspected in sporadic cases of alopecia in captive marine mammals but has not been demonstrated in wild populations (Gulland et al. 2001b).

The causes of several other dermatological syndromes reported in pinnipeds are unknown. Patchy to extensive alopecia and ulcerative skin lesions are frequently observed in northern elephant seals (Mirounga angustirostris) and although the syndrome has been extensively investigated, its etiology remains obscure (Beckmen et al. 1997; Yochem et al. 2008). Patchy alopecia is observed in Steiler sea lions (Eumetopias jubatus) and California sea lions (Zalophus californianus) in western North American waters and extensive alopecia is seen in gray seals (Halichoerus grypus) around the Farne Isles, United Kingdom (Gulland et al. 2001a). The cause of these syndromes of alopecia and their prevalence within these populations are unknown. The present study investigates a distinctive condition of alopecia in Australian fur seals. Its aims are to describe the histological appearance of alopecie skin and elucidate the pathologic mechanism and possible causal agents of this syndrome.

Materials and Methods

Animals.—Between September 2007 and February 2010, 11 visits, spaced approximately 3 months apart, were made to Lady Julia Percy Island (38°42′S, 142°00′E), in Bass Strait, Australia. On each visit, both alopecie (case) and unaffected (control) seals were captured for examination and diagnostic sampling. During each field trip, we aimed to pair cases with controls, for age class and sex. Animals were captured, restrained, and allocated to an age class by methods previously described (Lynch et al. 2011). All methods pertaining to live-animal handling were conducted in accordance with the guidelines of the American Society of Mammalogists (Sikes et al. 2011) and received approval from the Deakin University, Animal Welfare Committee.

Sample collection and analysis.—The pelage of each seal was visually examined for ectoparasites by combing the hairs of the dorsal and lateral body surfaces backward with the side of a hand. In addition, superficial and deep skin scrapings and adhesive tape preparations were collected. Superficial scrapings involved sweeping a scalpel blade coated in paraffin oil multiple times across a wide area of the side and back of the animal. Deep skin scrapes were performed by clipping the hair in a 9-cm2 area of the dorsum as short as possible and vigorously scraping with an oil-coated blade. Paraffin wet mounts were prepared from each scraping and examined by light microscopy in the field. Adhesive tape preparations were made by separating the back hair in multiple areas and briefly applying the adhesive surface to collect parasites present in the hair shafts or on the skin surface. The tape was mounted on a glass slide and examined under light microscopy. Skin scrapes and tape preparations were collected in the austral spring and summer because ectoparasitic infections of mammals are usually more prevalent in warmer months.

A single full-thickness skin biopsy was collected from both the thorax and rump of each seal using an 8-mm biopsy punch (Stiefel Laboratories Pty. Ltd., Melbourne, Australia). Biopsies were fixed in 10% buffered formalin. In case seals, the thoracic biopsy was collected from the edges of the area of alopecia to maximize the likelihood of demonstrating active and recent pathology. In several cases, samples also were collected from the lesion center. The biopsy sites were prepared by clipping hair to short stubble and lightly wiping the area with 70% alcohol. Following collection, sites were plugged for hemastasis with a calcium-sodium alginate wound dressing (Kaltostat; ConvaTec, Skillman, New Jersey). After formalin fixation, biopsies were embedded in paraffin, sectioned, and stained with hemotoxylin and eosin. Sections were examined under light microscopy to determine if histological lesions were present and to record the proportion of hair follicles that contained hair shafts.

Hair plucks (trichograms) comprising at least 20 guard hairs were collected from the dorsal thorax and rump of each seal. For cases, the thoracic sample was taken from the edges of the lesion and the rump sample from an unaffected area, to provide an intra-animal control. The hair bundle was laid on clear adhesive tape, attached to a glass slide, and examined by light microscopy. Hair tips were classified as either tapered or frayed-fractured if the tip was split or shaft fracture was evident (Fig. 2). Additional plucked hair also was used to inoculate dermatophyte test media (Fungassay; Synbiotics Corporation, San Diego, California). Inoculated media was monitored for color change that would indicate the presence of common dermatophytes. Fungal colonies that grew within 21 days were submitted to a diagnostic laboratory (Grabbles Veterinary Pathology, Glenside, South Australia) and identified to genus level by morphological features under KOH staining.

Fig. 2

Scanning electron microscope images of normal and frayed tips of guard hairs from Australian fur seals. Alopecie seals had higher proportions of damaged hairs than did unaffected seals.

Thoracic hair plucks (n = 10; cases = 5, controls = 5) and thoracic skin biopsies (n = 4; cases = 2 and controls = 2) were prepared for scanning electron microscopy. All samples were washed twice in absolute ethanol then suspended in a 1:1 dilution of absolute alcohol and hexamethyl-disilazone (ProS-ciTech, Thuingowa, Australia). Samples were then transferred to 100% hexamethyl-disilazone, which was allowed to evaporate. They were then mounted on stubs with double-sided adhesive tape and coated with gold in a Polaron E5000 sputter coater (Quorum Technologies, Ashford, United Kingdom). Specimens were examined using a Philips 505 scanning electron microscope (Philips Research, Eindhoven, The Netherlands).

Trace element concentrations were determined in blood and hair samples. Blood was collected from the brachial vein into trace element tubes (BD Vacutainers; Becton Dickinson and Company, North Ryde, Australia) and whole blood and plasma were harvested and stored in separate cryovials (Nalgene Thermo, Fisher Scientific Australia, Scoresby, Australia). These were held in liquid nitrogen for the duration of each field trip and then stored at − 70°C for up to 12 months. A 3-g mixture of thoracic and rump guard hair and underfur was clipped using sterile scissors from each seal and stored at ambient temperature for the duration of the field trip, and then at − 70°C prior to analysis. Trace element analysis of both blood and hair was performed at the Royal Prince Alfred Hospital (Camperdown, Australia), using inductively coupled plasma-mass spectroscopy. The analysis was performed using methods previously applied to pinnipeds (Gray et al. 2008). Trace elements measured were As, Cd, Hg, and Pb (hair and whole blood); Ni, Cu, Zn, Se, Cr, Fe, Al, and Co (hair and plasma); and Mg, Mn, Sn, Sb, and Tl (hair only).

Subcutaneous fat collected from the thoracic skin biopsy site was stored in cryovials at — 70°C. Analysis for 22 polychlori-nated biphenyl (PCB) congeners and a range of organochlorine compounds was performed by gas chromatography-mass spectrometry. Organochlorines included in the analysis were dichlorodiphenyltrichloroethane (DDT) and its metabolites, the cyclodienes (aldrin, dieldrin and endrin, heptachlor, chlordane, and endosulfan), the hexachlorocyclohexane isomers, hexa-chlorobenzene, methoxychlor, and mirex. Samples were between 80 and 160 mg wet-weight, resulting in a lower detection limit of 0.030 μg/g. Analysis was conducted according to the manufacturers' methods (Sandy 2009) at the laboratories of Advanced Analytical Australia Pty. Ltd. (North Ryde, Australia), using an Agilent 7890A GC (Agilent Technologies Ltd., Berkshire, United Kingdom).

Plucked guard hairs from the thoracic region were submitted to CSIRO Materials Science and Engineering (Parkville, Australia) for amino acid analysis by high pressure liquid chromatography (Spackman et al. 1958). The analysis utilized a Waters Alliance HPLC (Waters Ltd., Milford, Massachusetts) controlled by Enpower software (Enpower Software Ltd., Auckland, New Zealand). Amino acids assayed were cysteine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline.

Statistical analysis.—Statistical analyses were performed using the software program PASW Statistics 18 (SPSS Inc., Chicago, Illinois). Kolmogorov-Smirnov tests were applied to check if data were normally distributed and P > 0.05 was used to indicate normality. Independent t-tests were used to compare the proportion of follicles with guard hairs in skin sections from cases and controls. The respective influence of the categorical variables, case status, and months postmolt on the proportion of guard hairs fractured-frayed in thoracic trichograms was explored using 2-way analyses of variance. Trace metals in the hair and blood of juvenile female cases and controls were compared using independent t-tests for normally distributed parameters and Mann-Whitney U-tests otherwise. Hg concentration of blood was converted from nmol/liter to μg/liter using a conversion factor of 0.20059 (molar mass Hg ÷ 1,000). The relative percentages of each of the 16 amino acids assayed were calculated for each hair sample and compared for cases and controls using independent t-tests. PCB congeners and DDT metabolites were summed and compared between cases and controls by the Mann-Whitney U-test. Samples falling below detection limits were assigned a zero value. Relationships between parameters measured in blood and fat that showed significant differences between case and control groups were explored. Scatter plots were constructed and if relationships were linear, a Pearson product-moment correlation coefficient (r) was calculated. Similarly, the correlation between hair amino acids and hair metals was explored for those parameters that showed significant differences between case and control groups.

Results

Fifty-nine randomly selected alopecie seals were captured for diagnostic sampling, 55 juveniles (51 females and 4 males) and 4 adult females. Fifty-eight control animals also were sampled, 52 juveniles (39 females and 13 males) and 6 adult females. Six juvenile cases and 1 juvenile control captured for sampling exhibited ulceration of the skin of the ventral mandible (chin), suggestive of excessive rubbing to this area. In severe cases the lower lip was completely eroded and the surface of the exposed mandible abraded. In addition, 1.5% (6 of 406) of alopecie seals observed during field operations appeared to be intensely pruritic as evidenced by persistent (>5 min) and vigorous rubbing of the neck and chin on rocks. These distinctive behaviors were not observed in a non-alopecic seals.

Visual examination, superficial and deep skin scrapings (7 cases and 10 controls), and adhesive tape preparations (13 cases and 14 controls) did not detect ectoparasites on either case or control seals. Additionally, no evidence of deep ectoparasitic infestation was noted in any of the skin biopsies from 27 cases and 26 controls.

A change in color indicating growth of common dermatophytes was not observed in inoculated test media containing seal hair (9 cases and 10 controls). Of these samples, fungal colonies grown from the hair of 4 case and 4 control seals were identified as likely soil and plant saprophytes from the genera Cladophialophora, Acremonium, Alternaria, Penicillium, Paecilomyces, Chrysosporium, and Beauveria. Light microscopic examination of trichograms (n = 73) and skin sections (n = 53) did not reveal any fungal infection of skin or hair in either group.

Light microscopy examination of thoracic skin biopsies from cases and controls did not demonstrate histological differences. Both groups had an epidermal thickness of approximately 58 μm, with a stratum corneum of about 14 μm. The dermal layer comprised a connective tissue matrix containing the pilosebaceous unit (follicle, root and shaft of hair, and sebaceous glands), apocrine sweat glands, smooth muscle fibers, and cellular elements (fibroblasts, macrophages, and mast cells). Typically, the compound hair follicles consisted of a single guard hair and 22–27 secondary hairs (underfur). All animals had mild, perivascular, mononuclear infiltrates of the dermis but these were not associated with more widespread inflammation. No parasites, viral inclusions, or fungal hyphae were seen in histological sections from cases or controls.

In thoracic skin sections, there was no difference between cases and controls in the proportion of follicles containing guard hairs (t47 = −0.475, ̄case = 84%, ̄control = 85%, P = 0.637). For cases, no difference was found in the proportion of follicles with guard hairs between affected (thorax) and normal (rump) body regions (t42 = —0.673, ̄thorax = 84%, ̄rump+ — 86%, P = 0.505). Scanning electron microscopy images of biopsies taken from the center of alopecie areas showed fractured guard hairs present among the underfur that were not evident to the naked eye (Fig. 3).

Fig. 3

Scanning electron microscope image of a hair cluster biopsy from the central part of an alopecie lesion. Guard hairs (flattened, thick hairs) are evident among the underfur.

Trichograms from 45 cases and 38 controls showed that for both groups, the proportion of thoracic guard hairs with frayed or fractured tips increased with time after the annual autumnal molt (Fig. 4). In seals affected by alopecia, however, the proportion of frayed or fractured thoracic hair was close to 100% by 4 months postmolt, whereas control seals showed a slower rate of damage. Compared with controls, cases had a significantly higher proportion of frayed or fractured guard hairs on both thorax (F = 32.3, n = 83, P < 0.0005) and rump (F = 5.7, n = 83, P = 0.020). Cases also had a significantly greater proportion of frayed or fractured hairs on the thorax than the rump (F = 5.7, n = 45, P = 0.019), but there was no difference between these sites on control seals (F = 1.5, n = 38, P = 0.219).

Fig. 4

Percentage of frayed and fractured guard hairs (± SD) versus months postmolt that were identified in trichograms collected from the dorsal thorax of case (alopecie) and control (unaffected) Australian fur seals.

The hair of juvenile female cases had significantly higher levels of Mg, Co, Pb, and Ni and significantly lower Zn compared to juvenile female controls (Table 1). Case animals also had a significantly higher concentration of total Hg (tHg) in their blood than did controls. Mean hair Hg concentration for case and control juvenile females did not differ significantly (P = 0.281) and combined they averaged 14.7 μg/g (n = 35; SE = 2.7 μg/g, 95% confidence interval, 9.21–20.2).

View this table:
Table 1

Mean concentration ± SDof trace metals, ΣDDT, and ΣPCB in alopecie (case) and unaffected (control) juvenile, female Australian fur seals from Lady Julia Percy Island. Only parameters with significant differences between these groups are presented.

ParameterSiteaTest statisticCasesControls
MgHairt= 2.09, P = 0.0451,333 ± 417 (n = 21)1,059 ± 319 (n = 14)
CoHairt = 2.52, P = 0.0170.039 ± 0.017 (n = 21)0.025 ± 0.015 (n = 14)
ZnHairt = -2.82, P = 0.008150 ± 14 (n = 21)162 ± 11 (n= 14)
PbHairt= 2.46, P = 0.0190.171 ± 0.038 (n = 21)0.138 ± 0.041 (n= 14)
NiHairU = 231, P = 0.0051.219 ± 0.548 (n = 21)0.732 ± 0.194 (n = 14)
HgBloodt= 2.92, P = 0.015345 ± 87 (n= 6)215 ± 66 (n = 6)
ΣddtFatU = 135, P = 0.0320.273 ± 0.266 (n= 14)0.155 ± 0.148 (n = 13)
ΣPCBFatU = 149, P = 0.0040.095 ± 0.103 (n = 14)0.042 ± 0.081 (n = 13)
  • a Hair: μg/g dry weight; blood: μg/liter wet-weight; fat: μg/g wet-weight.

In fat samples from juvenile females (14 cases and 13 controls), many of the PCB congener and organochlorine values were below detection limits. However, case seals had significantly higher ΣDDT and ΣPCBs than control seals (Table 1). Blood Hg level positively correlated with fat ΣDDT (r - 0.71, n = 9, P = 0.031) and ΣPCB (r = 0.86, n = 9, P = 0.003) levels.

Compared to control seals, the guard hairs of juvenile female cases had lower percentage tyrosine (t20 = −2.907, ̄ ± SD, ̄cases = 0.69% ± 0.10%, ̄controls = 0.89% ± 0.19%, P = 0.009) and marginally higher percentage threonine (t20 = 2.166, ̄cases = 7.18% ± 0.20%, ̄controls = 6.87% ± 0.24%, P = 0.044). Tyrosine was significantly negatively correlated with Ni levels (r = −0.64, n = 17, P = 0.006) but did not correlate with Mg, Co, Zn, or Pb levels. No correlation was found between levels of threonine and the metals Mg, Co, Zn, Ni, or Pb.

Discussion

Mechanism of alopecia.—A key finding of this study is that the observed alopecia is caused by hair fracture above the level of the skin rather than loss from the follicle with the dermis. This was evident in scanning electron microscopy images of lesions where fractured guard hairs were still apparent, and in the lack of a statistically significant difference in the numbers of hair shafts present in follicles between skin sections from case and control seals. In all mammals, hair of normal composition and strength will fray and break if subjected to vigorous trauma. Most commonly, this is due to self-trauma where inflamed, pruritic skin is rubbed and scratched excessively causing alopecia (Hawryluk and English 2009; Scott and Paradis 1990). If, however, hair is of suboptimal quality, even mild trauma related to normal daily activities may fracture hair shafts (Cheng et al. 2009). Subsequent analysis in this study sought to determine the presence of possible causal factors for hair breakage in Australian fur seals and if these related to excessive self-trauma or suboptimal hair quality.

Lice infestations have been reported from pinnipeds worldwide, and can be associated with pruritis and alopecia (Dailey 2001). Lice, however, are easily observed by the naked eye (Mcintosh and Murray 2007; Thompson et al. 1998) and were not found on any of the >100 Australian fur seals examined in this study. Mite infestations of pinnipeds may be located on or beneath the skin surface or within the hair follicle and may cause pruritis (Dailey 2001; Dailey and Nutting 1980). The negative results obtained from skin and hair scrapings, and histological sections indicate that mites are an unlikely causal factor for this alopecia syndrome in Australian fur seals. The absence of inflammatory histopathological changes normally associated with viral and bacterial infections of the dermis suggest these types of pathogens also are unlikely causative factors for this syndrome. The results of microbiological and histological investigations also preclude fungal infections of the hair shafts or dermis as a likely causative factor in the alopecia syndrome.

Pruritis and self-trauma resulting in hair breakage also can be caused by immune-mediated diseases that produce hypersensitivity and skin inflammation (Scott and Paradis 1990). The observations of case seals grooming excessively or rubbing their body against rocks, and the severe ulceration of the skin of the ventral mandible of some individuals suggest these individuals were pruritic. However, seals were not observed excessively rubbing the site of hair loss and only a small proportion of cases were observed to be pruritic. In addition, pruritic animals would be expected to have obvious inflammatory cell infiltration of their dermis (Yager and Scott 1991) and this was not noted on skin biopsies from affected seals.

Structural and compositional differences in hair.—Trichogram analysis and hair compositional data indicate that differences in hair structure that may relate to hair strength are apparent between affected and unaffected Australian fur seals. The statistically significant higher proportion of damaged thoracic and rump guard hairs in alopecie seals suggests abnormally rapid degeneration following the molt. In addition, that the proportion of damaged hair in case seals was greater on the thorax than on the rump (with no difference in controls), is consistent with the thoracic region being the primary site of alopecia in sampled animals.

Hair fiber strength and integrity depends on its biochemical composition, a normal structure, and adequate protection by secretions from cutaneous glands (Jones 2001; Meyer et al. 2003). In Australian fur seals, juvenile females with alopecia had a statistically significant lower proportion of the amino acid tyrosine than controls, and this could theoretically result in lower fiber strength. Hair fibers consist of 3 main groups of proteins that are necessary for strength; high-tyrosine proteins and low- and high-sulfur proteins (Wu et al. 2008). A reduction in high-tyrosine proteins in sheep and mice is associated with weakened hair fibers (Gillespie et al. 1980; Reis and Gillespie 1985) and their deficiency in hair may result from altered protein intake, absorption, or metabolism (Reis 1992). To further investigate the significance of less high-tyrosine proteins in Australian fur seals would require knowledge of diets of cases and controls, and a greater understanding of key elements that control formation of keratin proteins in pinnipeds. Reduced content of the sulfurrich amino acids, cysteine and methionine, in hair also can cause increased fiber brittleness, because disulfide bonds lend strength to keratin (Wolfram 2003). In the Australian fur seals sampled, however, the cysteine proportion did not vary between cases and controls, whereas methionine levels were unable to be analyzed in this study. Comparison of the disulfide content of hair from case and control groups would be a worthwhile analysis to further investigate links between amino acid composition and alopecia.

In addition to low levels of tyrosine, juvenile female Australian fur seals with alopecia also had low concentrations of Zn in their hair. The trace element content of hair depends on the concentration of the elements in the blood during the period of hair growth (Kempson and Lombi 2011). In turn, blood trace metal concentrations are influenced by dietary intake as well as interactions with other elements and compounds (Kempson and Lombi 2011). Adequate Zn is essential for the production of normal hair, and a deficiency results in abnormal keratinization and weakened hair fibers in many species (Colombini 1999; Goldberg and Lenzy 2010; Reis 1992). The significantly lower Zn in case juveniles may indicate deficiency and, therefore, lower hair strength. The magnitude of the difference between groups, however, is small. Furthermore, normal hair Zn values are not well established for fur seals because most reported levels are for phocid species. A sample of 20 northern fur seals (Callorhinus ursinus) of varying ages had a mean hair Zn of 186 μg/g (Ikemoto et al. 2004). In Antarctic phocid seals, the mean hair Zn values include 137 ug/g in Weddell seals (Leptonychotes weddelliiGray et al. 2008) and 164 μg/g in juvenile southern elephant seals (Mirounga leoninaAndrade et al. 2007). In Northern Hemisphere phocids, adult Baikal (Pusa sibirica) and Caspian (Pusa caspica) seals had mean hair Zn values of 105 and 98 μg/g, respectively (Ikemoto et al. 2004). The broad interspecies variation in hair Zn values indicates that analysis of a greater number of hair samples from cases and controls from Lady Julia Percy Island is warranted. In addition, data for individuals from other Australian fur seal breeding colonies would help clarify the significance of the findings of the present study.

The hair of alopecie Australian fur seals had higher concentrations of Mg, Co, and Ni than controls. Excess dietary Mg and Co can be detrimental to the health of domestic animals, although dermatological issues are not a primary sign of their toxicity (Puls 1994). Nickel toxicity, however, commonly manifests in humans as an allergic dermatitis (Zhao et al. 2009). The toxic effects of Ni vary between species, however, and in domestic livestock excess Ni characteristically results in general debilitation (Puls 1994). In ringed seals (Pusa hispida saimensis), stillbirth was strongly associated with high Ni content (̄ = 13 μg/g) of fetal hair (Hyvärinen and Sipila 1984). Hair Ni, Mg, and Co concentrations measured in juvenile females from Lady Julia Percy Island are similar to those measured in phocids from Antarctic waters (Andrade et al. 2007; Gray et al. 2008), an environment expected to have low levels of anthropogenic pollutants. Therefore, the levels of these trace elements in Australian fur seals do not suggest toxicity. The observed differences between case and control hair for these elements likely reflect a difference in element homeostasis between the groups at the time of hair growth.

Toxic load differences.—Toxins may act directly on the hair follicle or can interfere with hair growth and strength by altering nutrient absorption or metabolism (Ibim et al. 1992; Lu et al. 2007). Alopecie Australian fur seals had a higher toxic load than controls, as indicated by the Pb content of hair, chlorinated pollutants in fat, and Hg in blood. The hair Pb concentration measured in cases was comparable to levels in Antarctic pinnipeds (Andrade et al. 2007; Gray et al. 2008), making it unlikely that the seals on Lady Julia Percy Island are being detrimentally impacted by this heavy metal. Similarly, the fat concentrations of ΣDDT and ΣPCBs are lower than those reported for numerous other marine mammal species (Evans 2003) but indicate greater toxin exposure of case seals.

In contrast, both case and control seals from Lady Julia Percy Island had blood and hair tHg levels that greatly exceed of those reported for most other marine mammal species (Beckmen et al. 2002; Brookens et al. 2007; Das et al. 2008; Gray et al. 2008; Ikemoto et al. 2004). The toxicity of Hg depends on its chemical type and the organic form methylHg (MeHg) is recognized for its severe toxicological effects (Kempson and Lombi 2011). In free-ranging marine mammals, the health effects of excessive MeHg are poorly understood but in vitro immunosuppressive effects have been demonstrated (Das et al. 2008; Lalancette et al. 2003). MeHg is the predominant form of Hg exposure in marine mammals, because inorganic Hg from soil, agricultural, and industrial runoff is converted to this form by aquatic saprophytes, and then bioaccumulates through trophic levels (Kempson and Lombi 2011). When consumed by marine mammals, though, MeHg is subject to detoxification so the proportion of tHg that is MeHg varies between tissue types (Wagemann et al. 1998). Despite the ongoing detoxification in some tissues, a high proportion of the tHg in marine mammal blood is in the form of MeHg (Lockhart et al. 1999; Stavros et al. 2008; Woshner et al. 2008). Therefore, the significantly higher tHg in the blood of alopecie seals would be expected to indicate that they also have greater blood concentrations of toxic MeHg than controls. In addition, the strong correlation between blood tHg and ΣDDT and ΣPCBs indicates that the exposure to these pollutants is likely to be linked, presumably contained in the same prey items or foraging area.

The possible health impacts of high Hg exposure in Australian fur seals are unknown. Moreover, although Hg exposure may cause allergic dermatitis in humans (Lerch and Bircher 2004), a direct relationship with alopecia has not been demonstrated previously in any mammal. One possible link may be that Hg in hair is largely in the form of MeHg-cysteine with Hg-S and Hg-C bonds (George et al. 2010). The disulfide bonds in hair are important for strength and therefore excessive Hg-S binding could potentially weaken the shaft structure, if it sequestered significant amounts of S. Mean levels of tHg in the hair of Lady Julia Percy Island juveniles (14.7 μg/g) were similar to those in adult Australian fur seals (12.7 μg/g) at the Seal Rocks colony (350 km east) in the 1980s (Bacher 1985). In that study, juveniles had hair Hg concentrations between 1.2 and 5.4 μg/g. Despite the higher concentrations measured in Lady Julia Percy juveniles, the lack of a statistically significant difference in hair tHg between alopecie and control seals suggests that Hg may not be a direct cause of hair loss, but it does signal high a higher exposure to pollutants.

Alopecia in Australian fur seals has the features of many endocrine skin diseases, bilateral symmetry and age and sex biases (Baker 1986; Hawryluk and English 2009). Endocrine disorders, however, usually cause alopecia by suppression of hair follicle activity, meaning hair of normal strength is not replaced when it is naturally shed (Hawryluk and English 2009). Therefore, the finding of this study that alopecia is due to hair breakage is not typical of an endocrine-linked mechanism. The finding of higher toxin levels in alopecie seals, however, warrants consideration as to whether one or a suite of toxicants could be acting as endocrine-disruptive compounds. These are compounds that can mimic or antagonize the effects of natural hormones, including the sex hormones. Detrimental effects from such compounds have been demonstrated in a wide range of species including marine animals (Hotchkiss et al. 2008; Routti et al. 2010).

Higher toxin concentrations in case seals indicate consumption of prey items with higher levels of pollutants. Heavymetal loads in pinnipeds are known to vary with the trophic level of prey they consume (Das et al. 2003a; Loseto et al. 2008). In addition, it is well recognized that proximity to the coast results in higher pollutant loads in marine mammals due to runoff from industrial and agricultural enterprises (Das et al. 2003b; Lavery et al. 2008; Savinov et al. 2011). The relationship between toxin load, foraging area, and prey type of alopecie and nonalopecic Australian fur seals is at present unclear. Satellite tracking of nonalopecic seals from Lady Julia Percy Island clearly suggests that juveniles tend to forage closer to the coast than do adults, and that female juveniles remain closer to the colony than do male juveniles (R. Kirkwood, pers. obs.). Dietary studies to determine the trophic status of alopecie versus control seals and the collection of foraging location data from alopecie animals may elucidate the role of foraging strategy in presentation of this condition.

This study establishes that infectious and parasitic causes of alopecia are unlikely to be involved in a potentially population-regulating alopecia syndrome in Australian fur seals. Also, it finds that alopecia is due to hair fracture above the skin rather than hair loss from the follicle. Compositional differences were found between the hair of affected and unaffected animals that can relate to hair strength. Furthermore, animals affected by alopecia have a greater toxic load than unaffected animals, which may reflect different foraging sites or prey selection. Comparisons of trace elements and pollutants between seals at Lady Julia Percy Island and other colonies are needed to clarify implications of concentrations carried by the Lady Julia Percy seals. No causal agent was found that would explain the distinctive bilateral symmetry and bias toward juvenile females of the alopecia syndrome.

Acknowledgments

Research was conducted under Department of Sustainability and Environment (Victoria) research permit 10004150. Funding was provided by Phillip Island Nature Parks; Melbourne Zoo; Department of Agriculture, Fisheries and Forestry (Canberra); and the Winifred Violet Scott Trust. We gratefully acknowledge all field volunteers— A. Mitchell, M. Wills, S. Murphy, D. Dyson, N. Heafield, K. Halloran, S. Dower, K. Scanlon, A. Hoskins, N. Schumann, J. Back, A. Howard, B. Oke, W. Eisner, J. Bergfeld, I. Hoffman, M. Terkildsen, and D. Stokeld. Thanks also to L. Barnett of the Melbourne Veterinary Specialist Centre and P. Wynne for their help with the project. Special thanks are extended to R. McQuilty of the Trace and Toxic Element Unit, Royal Prince Alfred Hospital, for making the trace element analysis possible.

Footnotes

  • Associate Editor was I. Suzanne Prange.

Literature Cited

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