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1 Department of Microbiology and Immunology, Vanderbilt University, A5301 Medical Center North, Nashville, Tennessee 37232, USA
2 Department of Integrative Physiology, University of Colorado, Boulder, Colorado 80309, USA
3 Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901-6501, USA
4 Corresponding author (email: douglas.c.woodhams{at}vanderbilt.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The global trend in amphibian population declines (Stuart et al., 2004) is demonstrated regionally in Central America. Although it is not the only agent of declines in this region, the southeastward spread of chytridiomycosis is depleting amphibian populations (Daszak et al., 2003; Lips et al., 2003a). Monitored populations have declined throughout Costa Rica (Pounds et al., 1997; Lips, 1998) and western Panama, and chytridiomycosis was associated with these events (Berger et al., 1998; Lips, 1999; Lips et al., 2003a).
Little is known about the factors that affect disease resistance or susceptibility in amphibians. Although there is an effort to predict the probability of population decline of species within the rainforest amphibian assemblage in Panama based on ecologic characteristics (Lips et al., 2003b), no studies have yet investigated the immune defenses of these species. Defense of the skin is critical for protection from many amphibian pathogens, including Aeromonas hydrophila, parasitic water molds (reviewed in Pessier, 2002; Rollins-Smith et al., 2002a), and Ribeiroia ondatrae (Johnson et al., 2001, 2002). Batrachochytrium dendrobatidis colonizes keratinized epithelium (Berger et al., 1998; Longcore et al., 1999; Pessier et al., 1999). Defense of amphibian skin involves adaptive and innate immune defenses (Carey et al., 1999). The skin of all vertebrates can develop adaptive immune responses. That is, antigen detected by antigen-presenting cells in the skin can activate both cell-mediated (T cell) and humoral (B cell) defenses. Lack of lymphocytic infiltration in the chytrid-infected skin of frogs suggests that these animals have a poor cell-mediated immune response against B. dendrobatidis (Berger et al., 1998; Pessier et al., 1999), and there is currently no evidence for a humoral defense. Innate defenses such as epithelial barriers, phagocytic cells, and antimicrobial peptides may be vital.
Recent evidence suggests that antimicrobial peptides may be important defenses against B. dendrobatidis infection. In vitro studies showed that many isolated antimicrobial peptides of frogs had potent activity against the fungal pathogen (Rollins-Smith and Conlon, 2005). Natural mixtures of skin peptides are also active (Rollins-Smith et al., 2002b; Woodhams et al., 2006), and this may be more relevant to the natural immune defense of amphibians given that some peptides may act in synergy to inhibit pathogen growth (Westerhoff et al., 1995; Rollins-Smith et al., 2002a). Anuran species show significant variation in the number, structure, and antimicrobial activity of skin peptides (Vanhoye et al., 2003; Apponyi et al., 2004; Conlon et al., 2004).
Susceptibility to disease may be partly controlled by immune defenses that vary among individuals and species. Our aim was to develop an index of predicted resistance to chytridiomycosis. In forming this index, we included measures of quantity of skin peptides produced and their potency against B. dendrobatidis in vitro. This index may be applied to predict the relative disease susceptibility of species within an assemblage. There are few other predictive tools to indicate the risk of disease emergence.
Some species may have strong peptide defenses and unique skin peptide profiles or "fingerprints." Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is a rapid method for obtaining a profile or "fingerprint" of peptides secreted from amphibian skin (Conlon et al., 2004). In addition to differentiation of species, skin peptide profiles can reveal variation within species and indicate evolutionary relationships (Apponyi et al., 2004). Individual variation in the peptide profile may indicate novel protective peptides that correspond to antimicrobial potency. Identifying these protective peptides in other species may also improve disease-risk prediction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Skin peptides were sampled in January 2004 from adult amphibians of nine species in the rainforest of Omar Torrijos H. National Park, El Copé, Coclé, Panama (8°40'N, 80°37'W) at an elevation of approximately 850 m. Amphibian populations at this site have been monitored since 1997 (Lips et al., 2003b). The species sampled included anurans Atelopus varius (n = 8), Centrolene ilex (n = 5), Centrolene prosoblepon (n = 7), Colostethus panamensis (n = 12), Eleutherodactylus punctariolus (n = 5), Phyllomedusa lemur (n = 9), and Rana warszewitschii (n = 1); and urodeles Bolitoglossa colonnea (n = 2) and Bolitoglossa schizodactyla (n = 10). Monitoring for the presence of B. dendrobatidis is ongoing and consists of swabbing the ventral surface or drink patch, thighs, and toe webbing 10 times each with sterile toothpicks (two per individual) and preserving them in 70% ethanol. Batrachochytrium dendrobatidis was not detected on over 800 individual amphibians sampled before August 2004 using polymerase chain reaction (PCR) analysis (Pisces Molecular, Boulder, Colorado, USA; Lips, unpubl. data). As the amphibians sampled were not infected, the assemblage was thought to be naïve to the pathogen.
For comparison with Panamanian amphibians, we also sampled skin peptides from species with known susceptibility or resistance (Table 1
): Rana pipiens (BioCorporation, Alexandria, Minnesota, USA; n = 5 adults), Xenopus laevis (Xenopus I, Dexter, Michigan, USA; n = 5 adults), and Bufo boreas (Colorado Division of Wildlife, John W. Mumma Native Aquatic Species Restoration Facility, Alamosa, Colorado, USA; n = 10 newly metamorphosed). Survival of natural populations of R. pipiens and X. laevis infected with B. dendrobatidis (Green et al., 2002; Weldon et al., 2004; Ouellet et al. 2005) and resistance to experimental exposure (Rollins-Smith and Conlon, 2005; Carey, unpubl. data) demonstrate that these species are quite resistant to disease development. We found that some of the R. pipiens and X. laevis sampled in our laboratory colony carry a sublethal infection of B. dendrobatidis by PCR analysis of skin swabs (A. Hyatt, CSIRO, Australian Animal Health Laboratory, Geelong, Victoria, Australia). In contrast, B. boreas was highly susceptible to B. dendrobatidis in natural populations (Muths et al., 2003), and in exposure experiments (Blaustein et al., 2005). Although mortality rate depended on dose and time of exposure, 100% mortality was reached within 26 days after exposure of toads to 1,000 zoospores (Carey, unpubl. data).
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-adrenergic agents. Sufficient peptides were collected from salamanders (Bolitoglossa) without injection. All animals were placed into 50 ml of collecting buffer (50 mM sodium chloride, 25 mM sodium acetate, pH 7.0) (Nutkins and Williams, 1989), and they remained largely submerged for 10 min while skin secretions accumulated. Animals were then removed from the containers and released, and the buffer containing peptides was acidified to a final volume of 1% HCl (Sigma) to inactivate endogenous peptidases that might also have been secreted (Resnick et al., 1991; Steinborner et al., 1997). The acidified collection buffer with skin secretions was immediately passed over C-18 Sep-Pak cartridges (Waters Corporation, Milford, Massachusetts, USA). The Sep-Paks were stored in vials with 2–5 ml of 0.1% HCl. Peptides bound to the Sep-Paks were eluted with 70% acetonitrile, 29.9% water, and 0.1% trifluoroacetic acid (v/v/v) and concentrated to dryness by centrifugation under vacuum. Although the Sep-Pak procedure removes salts and other contaminants, deionized water may be superior to collection buffer for mass spectrometry analyses. The total concentration of skin peptides recovered after Sep-Pak purification was determined by Micro BCA Assay (Pierce, Rockford, Illinois, USA) following manufacturer’s instructions, except that bradykinin (RPPGFSPFR; Sigma) was used to establish a standard curve (Rollins-Smith et al., 2002b).
Culture and maintenance of B. dendrobatidis
Seven isolates of B. dendrobatidis (Table 2) were maintained by standard methods (Rollins-Smith et al. 2002a, b). A peptide mixture from R. pipiens was tested against each isolate at concentrations of 25, 50, 100, 150, 200, 250, 500, and 1,000 µg/ml through 7 days of growth (see below for method). Isolate JEL 310 (originating from Smilisca phaeota from Fortuna, Panama, isolated by P. Murphy) was used for assays of peptide activity for all species unless noted otherwise.
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To compare the relative immunologic resistance of each species based on in vitro growth inhibition assays, we developed an index of predicted resistance to B. dendrobatidis. The predicted resistance was calculated by multiplying the mean percent inhibition of pathogen growth at a standard peptide concentration of 50 µg/ml by the quantity of peptides produced per gram body weight (gbw) for each species. To test for differences among species in quantity of peptides collected per gbw (µg/gbw), growth inhibition of B. dendrobatidis (%), and predicted resistance Kruskal-Wallis tests were performed with SPSS v. 12 (SPSS Inc., Chicago, Illinois, USA). Species were classified as having the potential for either strong predicted resistance or weak predicted resistance with Hierarchical Cluster Analysis (a species classified as potentially strongly resistant had at least one individual within that cluster), selecting Squared Euclidean Distances to measure between points and Ward linkage to measure the distance between two clusters. The variables total peptides/gbw and percent growth inhibition were included in the analysis.
MALDI mass spectrometry of peptides
Mixtures of amphibian skin peptides were analyzed by MALDI MS. A PerSeptive Biosystems Voyager Elite mass spectrometer (Applied Biosystems, Foster City, California, USA) was operated in reflector, delayed extraction, and positive ion mode. A stainless steel 100 well plate was spotted with 0.5 µl sample and subsequently 0.5 µl matrix solution and allowed to air-dry. Samples of peptide mixtures were tested at 100 or 1,000 µg/ml if enough material was available. The matrix solution consisted of 10 mg/ml -cyano-4-hydroxycinnamic acid (Sigma), 60% volume of acetonitrile, and 40% volume of 1% trifluoroacetic acid. For external calibration the following peptide standards were applied: bradykinin fragment 1–7 (monoisotopic mass 757.4) and bovine insulin (average mass 5,734.5) (Sigma). Each sample was analyzed by averaging signals from 256 consecutive laser shots. The MS data was acquired in the m/z range 500 to 7,000, truncated at m/z 4,500 and baseline-corrected with Data Explorer v4.4 (Applied Biosystems). The peak values shown represent the monoisotopic mass, [M+H]+. A few signals may show secondary peaks 22 mass units greater than the primary peak and probably represent a peptide plus sodium adduct [M+Na]+. Spectra may also show peaks at 568.1 and 650.0 because of matrix or background.
Growth inhibition of B. dendrobatidis
Peptide mixtures diluted to a known concentration were assayed for their ability to inhibit growth of B. dendrobatidis as previously described (Rollins-Smith et al., 2002a, b). Insufficient material was available from most of the small amphibians sampled for the determination of minimal inhibitory concentration. Instead, the percent growth inhibition at a standard peptide concentration of 50 µg/ml was measured. This low concentration may more accurately reflect conditions experienced in nature, because high concentrations must be induced from amphibian skin artificially (Rollins-Smith et al., 2005). Approximately 5 x 104 zoospores in 50 µl of broth were plated in replicates of five in 96-well flat-bottomed microtiter plates (Costar, Corning, New York, USA) to which we added serial dilutions of the peptides. The pH of this growth medium was 6.5–7.0. The optical density at 490 nm was obtained daily for 7 days with an MRX Microplate Reader with Revelation software (Dynex Technologies, Inc., Chantilly, Virginia, USA) for each experimental well and for control wells containing broth but no peptides (positive control) or broth with heat-killed B. dendrobatidis (negative control).
Active zoospores were counted on a standard hemocytometer slide to examine the effects of peptide mixtures on the activity of zoospores through time. For example, zoospores from isolate 254 (from R. pipiens) were diluted in water (adjusted to pH 2.5 to match the peptide solution) and in a solution containing 50 µg/ml peptide (pH 2.5). On opposite sides of the hemocytometer were placed the peptide-zoospore mixture and the water-zoospore mixture. The active zoospores were counted from each side alternately and the time recorded at each count.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Skin peptide profiles obtained by MALDI MS demonstrated marked differences among species (Figs. 1, 2). Some Xenopus laevis mass signals matched previously described antimicrobial peptides from that species including PGLa (m/z 1,968) and magainin-1 and 2 (m/z 2,409, 2,466) (Fig. 1A; Rollins-Smith and Conlon, 2005). Similarly, some Rana pipiens mass signals matched previously described antimicrobial peptides of the brevinin 1 family (m/z 2,563, 2,577, and 2,593) and ranatuerin-2P (m/z 3,000) (Fig. 1B; Goraya et al., 2000). Thus, natural mixtures of peptides from some species analyzed here contained conventional antimicrobial peptides. Most Panamanian species demonstrated complex mixtures of 5–25 peptides. Some of these peptides will be sequenced in ongoing studies. Only minor variations in peptide profiles were evident within species. Clusters of peptides with masses similar to antimicrobials described in other species (i.e., patterns similar to the brevinin 1 family in Rana pipiens, Fig. 1B) are displayed in C. prosoblepon and two Bolitoglassa salamanders, but not in the two bufonids (Fig. 2).
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Among the 12 amphibian species sampled there were significant differences in the total quantity of peptides recovered (µg/g; Kruskal-Wallis test; 
211 = 62.12; P<0.001) and antifungal activity (% inhibition at 50 µg/ml; Kruskal-Wallis test; 211 = 51.45; P<0.001; Table 1
). B. boreas, A. varius, and E. punctariolus produced the smallest quantity of skin peptides (41–137 µg/gbw; Table 1). Phyllomedusa lemur and X. laevis produced the most copious peptide secretions (approximately 2,000 µg/gbw; Table 1). However, the most active peptide mixtures against B. dendrobatidis (percent inhibition at 50 µg/ml peptides; Table 1) were produced by B. boreas (59.2%), C. prosoblepon (36.9%), and X. laevis (32.5%). Thus, amphibians producing more copious skin secretions did not necessarily produce more effective peptides against B. dendrobatidis. For example, B. boreas peptides were highly potent at inhibiting B. dendrobatidis growth (Table 1) in a dose-dependent manner (Fig. 3). However, because so little total quantity of peptides is released from the skin, the predicted immunologic resistance to B. dendrobatidis was low (Fig. 4). The secretions of B. boreas were primarily composed of small peptides in the m/z range of 700–760 (Fig. 2E). At concentrations of 50 µg/ml, B. boreas peptides did not inhibit B. dendrobatidis (isolate JEL 275) zoospore activity for up to 40 min, and activity was comparable to that of a water control at a matching pH of 2.5. In contrast, 50 µg/ml R. pipiens peptides (pH 2.5) inhibited B. dendrobatidis (isolate JEL 254) zoospore activity completely on contact.
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The quantity and quality of skin peptide mixtures from each of 12 species were combined to predict immunologic resistance to B. dendrobatidis (Fig. 4). Predicted resistance was significantly different among the species (Kruskal-Wallis test; 211 = 39.89; P<0.001). A hierarchic cluster analysis divided the species into two classes. Those species with strong predicted resistance included X. laevis, C. prosoblepon, and P. lemur. Individuals of X. laevis and R. pipiens with known sublethal infections had relatively strong to moderate predicted resistance. Conversely, B. boreas had relatively weak predicted resistance and is a species known to be susceptible to infection and lethal disease (Fig. 4; Muths et al., 2003). The susceptibility of most Panamanian species is not known. On the basis of our index, we predict that C. prosoblepon and P. lemur will have a more robust skin peptide defense against chytridiomycosis than other species. However, variation in growth inhibition was high for C. prosoblepon (Table 1), and peptide defenses may be variable among individuals. A comparison of two MALDI MS profiles from C. prosoblepon individuals with 13.6% and 84.7% B. dendrobatidis growth inhibition shows relatively higher peaks of some peptides, particularly in the cluster around 2,651.9 Da, for the individual with greater antifungal activity (Fig. 2A, B). These peptides have not yet been described.
Antimicrobial peptide sensitivity of B. dendrobatidis isolates
A reference peptide mixture was obtained from a laboratory R. pipiens and tested against seven geographically different B. dendrobatidis isolates. The growth-inhibitory activity of peptide mixtures was not significantly different among chytrid isolates (analysis of variance; F6,7 = 1.006; P = 0.489; Table 2). The mean inhibition (% ±SE) was 47.5 ± 3.0 at a peptide concentration of 50 µg/ml. The minimal inhibitory concentration of the mixture was usually between 250 and 500 µg/ml (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The ability to predict disease susceptibility is an important step in the conservation of amphibians at risk of disease-associated population declines. Ecologic correlates of disease may provide valuable indicators of susceptibility (Lips et al., 2003b). These, in combination with indicators of host immunologic resistance, create a valuable tool for disease ecology and wildlife management. For example, species with potentially weak predicted defenses may be targeted for captive husbandry. However, the link between in vitro peptide effectiveness and protection in the wild is tentative (Woodhams et al., 2006).
Differences among species were found in both predicted resistance to chytridiomycosis and profiles of skin peptide mixtures examined by mass spectrometry. The index of predicted resistance to chytridiomycosis indicates weak levels of resistance for most species of Panamanian amphibians studied, similar to the predicted resistance of B. boreas, a species known to be susceptible.
We found that MALDI MS was a rapid method to obtain unique skin-peptide profiles, and can be used for phylogenetic comparisons. The bufonids (true toads) B. boreas and A. varius had similar skin peptide profiles characterized by a set of small peptides not typical of the larger antimicrobial peptides found in other species (Zasloff, 2002; Apponyi et al., 2004; Rollins-Smith and Conlon, 2005). Conlon et al. (2004) did not detect antimicrobial peptides in Bufo skin. Similarly, A. varius may lack conventional antimicrobial peptides. The small peptides found in these species may have alternative functions. We found that the peptide mixtures of B. boreas were active against B. dendrobatidis but did not completely kill the fungus. Although the B. boreas peptide mixtures inhibited fungal growth in vitro, the small quantities of peptides produced by the toads may not provide significant protection in vivo.
Phylogenetic constraints on innate immune defenses may help explain the taxon bias in declines found by the Global Amphibian Assessment (Stuart et al., 2004). For example, Bufonidae is one of four amphibian families that contain significantly more than the average number of rapidly declining species (Stuart et al., 2004). This taxon has a distinctively thick epidermis, often with a heavily keratinized and roughened structure (Elias and Shapiro, 1957), and skin peptide profiles of this family are notably less complex than in other taxa. These features may increase susceptibility to chytridiomycosis.
Only two Panamanian species, C. prosoblepon and P. lemur, were predicted to be more resistant than North American R. pipiens, which survives infection with B. dendrobatidis (Ouellet et al., 2005). MALDI MS indicated that potentially novel antimicrobial peptides are present in the skin-peptide mixtures of C. prosoblepon. The large variability in the growth inhibition activity of C. prosoblepon peptides may indicate that not all individuals are equally defended. Those individuals that are better defended may survive chytridiomycosis epizootics. We hypothesize that variation in skin peptide defense is reduced at sites of disease emergence where small populations of survivors persist. For example, at Fortuna, Panama, populations of many amphibian species declined in 1996 and chytridiomycosis is the suspected agent of declines (Lips, 1999), but small populations of C. prosoblepon persist. These surviving populations may have enhanced skin peptide defenses relative to populations that are naïve to this strong selection pressure.
Batrachochytrium dendrobatidis–resistant R. pipiens experienced population declines in the past, although the causes are not fully known (NatureServe, 2004). A preserved R. pipiens from Colorado, 1974, represents the earliest known infection of that species (Carey et al., 1999), and recent studies found infected frogs in apparently healthy wild populations (Green et al., 2002). Historic evidence suggests that B. dendrobatidis is enzootic in R. pipiens (Ouellet et al., 2005). The strength of X. laevis resistance is consistent with a coevolutionary history with the fungus. Studies of preserved museum specimens have found the earliest B. dendrobatidis infection in X. laevis from South Africa in 1938, and enzootic infections were found in nondeclining populations in South Africa (Weldon et al., 2004). Antimicrobial peptide defenses may evolve more readily than components of the adaptive immune system (Zasloff, 2002; Vanhoye et al., 2003). Rapid peptide variation may result from error-prone DNA polymerases (Vanhoye et al., 2003). As disease advances through Panama, there is the opportunity to study this strong selection pressure on the evolution of peptide immune defenses.
The disease resistance observed in R. pipiens and X. laevis stands in contrast to the sensitivity of C. panamensis and Eleutherodactylus cruentus. When frogs were brought into captivity after collection from El Copé, Panama, in September 2004, mass mortality resulted from naturally contracted chytridiomycosis, and wild populations later disappeared (Carey and Voyles, unpublished data). Many other Panamanian species are showing susceptibility to chytridiomycosis, the first signs of which appeared at El Copé in October 2004 (Lips et al., unpubl. data). Dead amphibians of species examined here were found positive for B. dendrobatidis. The extent of the declines of these populations is not yet known.
Comparison of B. dendrobatidis isolates
Piotrowski et al. (2004) compared the growth curves of B. dendrobatidis at various temperatures and found similar growth among isolates. We also found little variation among isolates in sensitivity to an amphibian skin peptide mixture. It is not known whether an isolate of B. dendrobatidis or any other organism can be artificially selected for resistance to an antimicrobial peptide. Although infection experiments comparing survival of amphibians after exposure to different B. dendrobatidis isolates are still needed, our results suggest a lack of adaptation to particular species or environments and therefore little genetic differentiation among isolates. Genetic comparisons of 35 B. dendrobatidis isolates from North America, Panama, Africa, and Australia demonstrate that all are closely related (Morehouse et al., 2003). This supports ecologic, pathologic, and biogeographic evidence that chytridiomycosis is an emerging infectious disease of amphibians (Daszak et al., 1999; Weldon et al., 2004). This also suggests that B. dendrobatidis is a generalist pathogen and that species possessing an innate immunologic defense at the time of emergence are more likely to survive.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Received for publication 2 August 2005.
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