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Comparison of serum protein electrophoresis values in wild and captive whooping cranes (Grus americana).

Abstract: Protein electrophoresis of serum samples from endangered, wild whooping cranes (Grus americana) was performed to help assess the health of the only self-sustaining, migratory population in North America. Serum samples from wild adult cranes (n = 22) were taken at Aransas National Wildlife Refuge, Texas, USA during winter. Wild juvenile cranes (n = 26) were sampled at Wood Buffalo National Park, Northwest Territories, Canada, in midsummer. All captive crane samples were acquired from the International Crane Foundation, Baraboo, WI, USA. Captive adult cranes (n = 30) were sampled during annual examinations, and archived serum samples from captive juvenile cranes (n = 19) were selected to match the estimated age of wild juveniles. Wild juveniles had significantly lower concentrations of all protein fractions than wild adults, except for prealbumin and [gamma] globulins. All protein fraction concentrations for wild juveniles were significantly lower compared with captive juveniles, except for prealbumin and y globulins, which were higher. Wild adults had significantly greater [gamma] globulin concentrations than captive adults. Captive juveniles had significantly lower prealbumin and albumin concentrations and albumin: globulin ratios than captive adults. The higher [gamma] globulin concentrations in wild versus captive cranes are likely because of increased antigenic exposure and immune stimulation. Protein fraction concentrations vary significantly with age and natural history in this species. Reference intervals for serum protein electrophoresis results from captive adult whooping cranes are provided in this study.

Key words: serum protein electrophoresis, reference interval, [gamma] globulins, avian, whooping crane, Grus americana

Introduction

Whooping cranes (Grus americana) are one of two extant crane species native to North America (Family Gruidae). The species is endangered, protected under the US Endangered Species Act, and listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. A single self-sustaining population migrates from breeding grounds in Wood Buffalo National Park, Northwest Territories, Canada, to winter at the Aransas National Wildlife Refuge, Texas, USA. Populations for captive breeding and reintroduction are also maintained at five breeding facilities in the United States and Canada. (1-3)

Because of the endangered status of the whooping crane, the wild population is remotely monitored through aerial surveillance. (3) Routine capture and diagnostic tests are not performed to assess health, as to not disrupt normal breeding and migration. (1,3-6) In contrast, diagnostic testing is regularly performed on captive whooping cranes to assess health status, particularly before reintroduction. (7) 9 The wild whooping crane population is still at risk from habitat loss, pollution, collision with power lines, predation, low genetic diversity, parasites, and disease. (1,10-12) Diseases of whooping cranes are well documented, and include campylobacteriosis, salmonellosis, colibacillosis, mycobacteriosis, aspergillosis, viral encephalitis, coccidiosis, trauma, pododermatitis, osteomyelitis, developmental limb abnormalities, degenerative musculoskeletal disease, exertional myopathy, egg binding and yolk coelomitis, and lead and zinc toxicosis, among others. (5,7-10,2,13) To assess cranes for diseases that can affect breeding and survival, physical examinations, complete blood counts, and biochemical profiles are commonly performed on captive cranes. (7,8)

Protein electrophoresis has been recommended as a standard part of screening tests for avian species to measure blood proteins. (14-19) Blood proteins, including albumin and globulins, are important measures of metabolism, inflammation, infection, clotting, and immune response. (20) Changes in protein levels with protein electrophoresis have been documented with disease processes such as chlamydophilosis, mycobacteriosis, aspergillosis, sarcocystosis, egg yolk coelomitis, hepatitis, nephritis, hypersensitivity reactions, myeloma, and lymphosarcoma in avian species.(14,15,18-28) Changes can also occur with physiologic changes such as egg laying. (20,21) Because certain diseases and alterations in physiology have been shown to alter protein fractions in avian species, protein electrophoresis is therefore an important diagnostic tool in assessing overall health. (22)

Protein electrophoresis is the gold standard for measuring albumin and other protein fractions in avian species. (20,29) Measures of total protein and albumin by biochemical methods and refractometer are known to be less reliable in avian species. (20,21,29,30) With protein electrophoresis, avian species have been shown to have six protein fractions: prealbumin, albumin, and [alpha]-1, [alpha]-2, [beta], and [gamma] globulins. (14,21,22,24,30,31) The presence of prealbumin is not consistent among studies in avian species but has been reported in pigeons, raptors, and psittacine birds. (14,21,22,25) The use of protein electrophoresis in avian species as a baseline diagnostic and prognostic test is expanding, and laboratories are improving the techniques used to assess the fractions. (14,17,19,32-34)

Unfortunately, there are few reference intervals in the literature for protein electrophoresis in avian species. Most published ranges are for psittacine species and were determined from plasma instead of serum. (14,23,33,35) Although plasma is routinely used in avian medicine for biochemical and electrophoresis analyses, the use of serum reduces the bias from fibrinogen that normally migrates with the [beta] globulins. (21,25) The only study to interpret protein electrophoresis in cranes was one that assessed the response to presumed insect bite hypersensitivity reactions. (19) However, this study used individually matched baseline data. not population-based values. (19) There are no published reference intervals for protein electrophoresis for any crane species, despite the diagnostic value of this test, the common representation of these species in managed captive collections, and the endangered status of many of these species. (1)

The purpose of this study was to measure protein electrophoresis values from serum samples of wild juvenile and adult whooping cranes and compare them with those from age-matched captive cranes. In addition, reference intervals for serum protein electrophoresis results from the captive adult whooping cranes were established in this study.

Materials and Methods

Samples

Serum samples from wild whooping cranes were obtained after capture for satellite telemetry and population health assessment projects under valid US Fish and Wildlife Service, Canadian Wildlife Service, and state and provincial permits. Sampling protocols were approved by a University of Wisconsin Institutional Animal Care and Use Committee. Prefledgling juvenile cranes (n = 26) were captured by hand at Wood Buffalo National Park approximately August 1, 2010-2012, following the methodology of Kuyt. (36) Adult cranes (n = 22) were captured at Aransas National Wildlife Refuge with a self-activated or remotely triggered leg snare followed by manual restraint within 30 seconds during winter between December 2009 and January 2013. All birds were hooded, and blood was collected from the right jugular vein. Blood was dispensed into microvials with no additive (Sarstedt, Niimbrecht, Germany), allowed to clot under refrigerated conditions, and spun with an IDEXX StatSpin centrifuge (IDEXX Laboratories, Westbrook, ME, USA). Aliquots of serum were placed into cryovials and stored at -20[degrees]C until return to the International Crane Foundation (2-14 days), where the samples were stored at -80[degrees]C until analysis.

Serum from captive whooping cranes was obtained from samples archived at -80[degrees]C at the International Crane Foundation in Baraboo, WI. USA. The captive cranes were part of either the adult breeding flock or juveniles being raised in captivity for eventual release into the wild. (7) The estimated age of wild prefledgling juveniles was 49-70 days old. based on tarsal length measurements, annual Canadian Wildlife Service nesting surveys, and historic nesting phenology. (37-39) Sampling of captive juveniles in this age range is not common because of the release management strategy. Therefore, serum samples from captive juveniles were included from a wider range of years and were designed to match the estimated age range of wild juveniles. Serum samples from captive juveniles (n = 19) were collected during physical examinations in July 2001 (n = 9), September 2007 (n = 1), September 2008 (n = 4), September 2009 (n = 1), September 2011 (n = 2), August 2012 (n = 1), and August 2013 (n = 1). Serum samples from adults (n = 30) were collected during the September-October 2012 flock health check and were also used for reference interval determination. All samples of captive origin were from clinically healthy individuals.

Serum protein electrophoresis

Serum samples were analyzed according to the procedure provided by the Helena SPIFE 3000 system and Split Beta gels (Helena Laboratories, Inc, Beaumont, TX, USA). Results were produced after gel scanning and densitometry analysis by Helena software (Helena). Percentages for each fraction were determined by this software, and absolute values (g/dL) for each fraction were obtained by multiplying the percentage by the total protein concentration. The albumin: globulin (A: G) ratio was calculated by dividing the sum of prealbumin and albumin by the sum of the globulin fractions.

Statistical analyses

Electrophoresis reports were visually inspected, and descriptive statistics determined for absolute (g/dL) protein fraction measures. Protein fraction values from three cranes (one wild adult, one wild juvenile, and one captive juvenile crane) were observed >3 SD from their respective group mean and were classified as outliers. The entire electrophoretic records for the 3 cranes were eliminated from additional statistical analysis. Remaining values were assessed for normality using the Anderson-Darling test (Q1 Macros for Excel 2013, Know Ware International, Inc, Denver, CO, USA). Comparisons were made between the wild and captive population by age class and within the wild and captive populations between age classes. Statistical differences in electrophoresis results and A: G ratios were analyzed by using either the Student's t test (normally distributed data) or the nonparametric Mann-Whitney test (Statview 5.0.1, SAS Institute, Inc, Cary, NC, USA). Statistical significance was established at P < .05. Protein fraction reference intervals were calculated for the captive adults (n = 30) only using Excel Freeware Reference Value Advisor (RVA) v2.1 (40) (National Veterinary School, Toulouse, France) in accordance with the American Society of Veterinary Clinical Pathology guidelines. (41,42) No outliers were identified by the Dixon-Reed and Tukey methods, all data were normally distributed, and reference intervals were calculated using the parametric method in the RVA software. Normal distribution was determined by examining histograms of the data and confirmed with the Anderson-Darling test in the RVA software.

Results

Forty-nine captive and 48 wild whooping cranes were analyzed. The signalments when known are shown in Table 1. The results of the serum protein electrophoresis by population and age class are shown in Table 2. An example electrophoretogram from an agarose gel is shown in Figure 1. Juvenile wild whooping cranes had significantly lower concentrations of total protein (P < .001), albumin (P < .001) and [alpha]-1 (P < .001), [alpha]-2 (P < .001), and [beta] globulins (P < .01), and a decreased A: G ratio (P < .001) when compared with wild adult whooping cranes. All protein concentrations for wild juveniles were significantly lower compared with those of captive juveniles (total protein P < .001, A : G ratio P < .05, albumin P < .001, [alpha]-1 P < .001, [alpha]-2 P < .01, [beta] P < .001), except for prealbumin (P < .001) and [gamma] globulins (P = .03), which were greater. Wild adult whooping cranes had significantly higher concentrations of [gamma] globulins than captive adult cranes, (P < .01). Captive juveniles had significantly lower prealbumin (P < .01), albumin (P < .05), and A:G ratios (P < .01) than captive adults. Table 3 provides reference intervals for serum protein electrophoresis results from the captive adult whooping cranes in this study.

Discussion

Protein electrophoresis is an important diagnostic test for avian species. When combined with a complete blood count and biochemical profile, results of protein electrophoresis may identify inflammation and aid in the diagnosis of certain diseases. (14,15,18) The purpose of this study was to assess serum protein electrophoresis values in wild juvenile and adult whooping cranes and compare them with age-matched captive cranes. The analysis of these data and generation of reference intervals for captive adult cranes may improve health assessments of both captive and wild whooping cranes.

Avian species have been shown to have six protein fractions in protein electrophoresis: prealbumin, albumin, and [alpha]-1, [alpha]-2, [beta], and [gamma] globulins. (14,21,22,24,30) In this study, whooping cranes had all six of the protein fractions identified through laboratory analysis. These findings differ from a previous study in cranes with type I hypersensitivity that was not able to differentiate prealbumin or separate [alpha]-1 and [alpha]-2 globulins. (19) Methodologies for serum protein electrophoresis in avian species have changed in recent years. (32,34) The aforementioned study used the Sebia hydragel system (Sebia Inc., Norcross, GA, USA) for the protein electrophoresis methodology, whereas this study used the Helena system (Helena) for its methodology. The use of different systems and laboratories likely resulted in fraction identification differences. (24,32,34) As the systems are not completely comparable, (32) comparisons between laboratories can be difficult and this fact should be kept in consideration when assessing new data. In addition, these values were generated from serum, and clinicians wishing to compare values from plasma should be aware that the [beta]-globulin concentrations may not be comparable because of the lack of fibrinogen in the serum samples, which will in turn alter the total protein value and the A: G ratio as well. (21,28)

In this study, juvenile wild whooping cranes had significantly lower total protein, albumin, [alpha]-1, [alpha]-2, and [beta] globulin concentrations and a decreased A: G ratio when compared with wild adults. Developmental differences in hematologic results and serum biochemical profiles between juveniles and adult birds have been documented in avian species. (8,35) Previous reports state that juvenile eclectus parrots (Eclectus roratus) and cockatoos (Cacatua species) had significant age-related variations in complete blood count and biochemical values. (43,44) Juvenile birds tend to have lower albumin and [gamma] globulin levels than adults. (35) A similar pattern was documented in juvenile macaws (Ara species) sampled at 30, 60, and 90 days. The juvenile macaws had significantly varied total protein, albumin, globulin, and prealbumin concentrations and A : G ratios, which were lowest at 30 days and increased with age. (35) This indicates that juvenile cranes should have separate reference intervals than adults because of the physiologic effects of growth and flegding. (35)

Captive juveniles and captive adults have fewer differences in values than wild juveniles and wild adults. Captive juveniles had significantly lower prealbumin and albumin concentrations and A : G ratios than the captive adults. The fewer differences among captive whooping cranes may be caused by the influence of artificial husbandry, including use of uniform, contained environments; a formulated, nutritionally complete diet; and regular dewormings. (4,8) The available serum samples were also closer in season than those collected from wild whooping cranes. Seasonal variation in metabolism may explain some of the differences observed in the wild whooping cranes.

In this study, wild juvenile and adult whooping cranes had significantly higher levels of [gamma] globulins than captive counterparts. The [gamma] globulins are immunoglobulins and consist mainly of IgY. (45) Elevated [gamma] globulin levels indicate a chronic inflammation or infection, as the bird's immune system is responding to antigenic stimulation. (16) The higher [gamma] globulin level in wild cranes is likely because of a greater antigenic exposure to potential infectious agents in the wild. Unfortunately, no studies that we are aware of compare protein electrophoresis values between free-ranging and captive avian species, but this pattern has been reported in a mammal species. (46) Free-ranging dolphins (Tursiops truncatus) had a significantly lower A : G ratio from a decreased albumin and a two- to threefold increase in the [gamma] globulin concentration when compared with captive dolphins. (46) In avian species, inflammatory conditions, infectious diseases, and neoplasia may lead to high [gamma] globulin levels. (16,21,22-28) The wild whooping cranes reported here were considered healthy based on findings on physical examinations in the field, and had normal serum biochemical profiles (data not shown), but unlike the captive cranes, additional follow-up was not possible. Some underlying disease may not have been identified.

Most protein concentrations for wild juveniles were significantly lower than those of captive juveniles, except for prealbumin and [gamma] globulin, which were significantly higher. The captive juveniles were aged-matched to the wild juveniles to reduce the variation in proteins caused by the metabolic demands and changes seen in preparation for fledging. (35) Age estimation for the wild juveniles of unknown hatch date was based on tarsal length measurements, annual nesting surveys, and historic nesting phenology. (37-39) The cause of the lower protein levels in the wild juveniles is uncertain but may reflect differences in metabolic demands due to varied environment, diet, exercise, parasite load, exposure to diseases, and predator pressures in the wild. (10,12)

To best age-match the wild juveniles, archived serum samples from captive juveniles were selected to include individuals that were 48-79 days old. Because of management practices of captive juvenile cranes, very few birds are sampled during this age range. Therefore, serum samples that had been stored up to 12 years were included to produce a useful sample size. Samples were properly stored at -80[degrees]C to minimize protein degradation. The lack of more recent age-matched samples and possible degradation of proteins over time is one potential drawback of use of these samples. Previous studies show that a single freeze-thaw cycle did not significantly change avian protein fractions (14); however, more than one freeze-thaw cycle may have occurred to some of the samples due to the division and aliquoting of the banked samples for needed research on this endangered species. The documented change in the A: G ratio in avian samples after 3 freeze-thaw cycles was not significant and minimally altered the ratio by <2.1 [+ or -] 1.0%. (14) To our knowledge, the captive juvenile samples were not refrigerated for an extended period before or during analysis. Refrigeration up to 7 days can significantly change the protein fractions. (14) Refrigeration significantly changes the [alpha]-2 band, causing it to be less distinct and more difficult to quantify by densitometry, which in turn leads to a decreased A : G ratio. (14) This does not appear to have occurred with this study's samples, as all wild juvenile samples had lower A : G ratios and [alpha]-2 concentrations than the captive juveniles. The captive juveniles did have lower A : G ratios than captive adults, but the [alpha]-2 concentrations were not significantly different and the lower A: G ratio is a previously reported developmental difference seen in other avian species. (35)

Protein fractions vary significantly with age and the natural history of the individual and should be taken into consideration when assessing results of serum protein electrophoresis from whooping cranes, and likely other crane species. Wild adult and juvenile whooping cranes had significantly higher levels of [gamma] globulins than age-matched captive cranes, which may indicate increased antigenic exposure and immune stimulation among wild cranes. In addition, juvenile wild and captive whooping cranes had significantly lower concentrations of many protein fractions when compared with adult cranes. Future research should include improved sample sizes among various age classes and from multiple facilities to develop refined reference intervals for serum protein electrophoresis in whooping cranes.

Acknowledgments: We especially thank members of the Whooping Crane Tracking Partnership for logistics and personnel involved in the safe capture of the wild cranes, as well as the staff and administration of Wood Buffalo National Park and the Aransas National Wildlife Refuge for their support. The Calgary Zoo veterinary staff assisted with sample transfer and permits. We also extend appreciation to the staff of the Crane Conservation and Veterinary Services departments of the International Crane Foundation. A. Krisp assisted with calculations of the reference intervals. This project was supported by the Companion Animal Fund of the University of Wisconsin and the Avian and Wildlife Laboratory, at the University of Miami.

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Table 1. Signalments where known of all whooping cranes from which
serum samples were analyzed by protein electrophoresis.

                     Captive juvenile      Captive adult

Age, mean (median)     66 (70) days     20 years (22 years)
Age, range             48-79 days       3M5 years
Male, n                 8               15
Female, n              11               15
Total, n               19               30

                     Wild juvenile   Wild adult

Age, mean (median)        --             --
Age, range                --             --
Male, n                   17             9
Female, n                  9             13
Total, n                  26             22

Table 2. Protein electrophoresis values from serum samples of wild
and captive whooping cranes compared by age group (juvenile and
adult).

                                   Captive juveniles (n = 19)

Analyte                    Mean [+ or -] SD (median)          Min-Max

Total protein, g/dL         3.4 [+ or -] 0.7 (3.6) (a)         2.0-4.8
Albumin: globulin ratio     1.1 [+ or -] 0.2 (1.) (a,c)        0.7-1.5
Prealbumin, g/dL           0.13 [+ or -] 0.04 (0.13) (a,c)    0.05-0.24
Albumin, g/dL              1.64 [+ or -] 0.32 (1.65) (a,c)    1.05-2.28
[alpha]-1 globulin, g/dL   0.18 [+ or -] 0.06 (0.16) (a)      0.09-0.31
[alpha]-2 globulin, g/dL   0.63 [+ or -] 0.18 (0.56) (a)      0.37-1.02
[beta] globulin, g/dL      0.55 [+ or -] 0.21 (0.50) (a)      0.25-1.02
[gamma] globulin, g/dL     0.32 [+ or -] 0.16 (0.34) (a)      0.08-0.59

                                   Captive adults (n = 30)

Analyte                    Mean [+ or -] SD (median)        Min-Max

Total protein, g/dL         3.7 [+ or -] 0.7 (3.8)           2.2-52
Albumin: globulin ratio     1.3 [+ or -] 0.2 (1.3) (c)       1.0-1.7
Prealbumin, g/dL           0.17 [+ or -] 0.05 (0.18) (c)    0.09-0.29
Albumin, g/dL              1.93 [+ or -] 0.44 (1.89) (c)    0.94-3.02
[alpha]-1 globulin, g/dL   0.18 [+ or -] 0.05 (0.17)        0.10-0.30
[alpha]-2 globulin, g/dL   0.59 [+ or -] 0.10 (0.60)        0.33-0.78
[beta] globulin, g/dL      0.54 [+ or -] 0.16 (0.52)        0.31-0.84
[gamma] globulin, g/dL     0.30 [+ or -] 0.06 (0.31) (d)    0.15-0.42

                                   Wild juveniles (n = 26)

Analyte                    Mean [+ or -] SD (median)          Min-Max

Total protein, g/dL         2.6 [+ or -] 0.5 (2.6) (a,b)       1.8-3.6
Albumin: globulin ratio     1.0 [+ or -] 0.2 (0.9) (a,b)       0.7-1.4
Prealbumin, g/dL           0.19 [+ or -] 0.05 (0.19) (a)      0.10-0.28
Albumin, g/dL              1.07 [+ or -] 0.26 (1.04) (a,b)    0.69-1.68
[alpha]-1 globulin, g/dL   0.10 [+ or -] 0.03 (0.10) (a,b)    0.06-0.17
[alpha]-2 globulin, g/dL   0.48 [+ or -] 0.11 (0.47) (a,b)    0.33-0.76
[beta] globulin, g/dL      0.36 [+ or -] 0.11 (0.33) (a,b)    0.18-0.64
[gamma] globulin, g/dL     0.40 [+ or -] 0.10 (0.40) (a)      0.28-0.62

                                   Wild adults (n = 22)

Analyte                    Mean [+ or -] SD (median)        Min-Max

Total protein, g/dL         3.6 [+ or -] 0.5 (3.6) (b)       2.8-4.6
Albumin: globulin ratio     1.3 [+ or -] 0.2 (1.2) (b)       1.0-1.7
Prealbumin, g/dL           0.19 [+ or -] 0.04 (0.17)        0.12-0.26
Albumin, g/dL              1.85 [+ or -] 0.35 (1.71) (b)    1.38-2.66
[alpha]-1 globulin, g/dL   0.17 [+ or -] 0.04 (0.17) (b)    0.11-0.26
[alpha]-2 globulin, g/dL   0.62 [+ or -] 0.11 (0.60) (b)    0.42-0.80
[beta] globulin, g/dL      0.46 [+ or -] 0.09 (0.48) (b)    0.27-0.62
[gamma] globulin, g/dL     0.36 [+ or -] 0.07 (0.37) (d)    0.24-0.47

(a) Wild juveniles statistically different than captive juvenile
cranes, P < .05.

(b) Wild adults statistically different than wild juvenile cranes,
P < .05.

(c) Captive juveniles statistically different than captive adult
cranes, P < .05.

(d) Wild adults statistically different than captive adult cranes,
P < .05.

Table 3. Reference intervals for serum protein electrophoresis values
from captive adult whooping cranes (n = 30).

                                          Lower          Upper
                           Reference    reference      reference
Analytes                   interval    limit 90% CI   limit 90% CI

Total protein, g/dL         2.3-5.1      2.0-2.7        4.8-5.5
Albumin: globulin ratio     0.9-1.7      0.8-1.0        1.6-1.8
Prealbumin, g/dL           0.06-0.29    0.04-0.09      0.26-0.32
Albumin, g/dL              1.02-2.85    0.76-1.27      2.60-3.11
[alpha]-1 globulin. g/dL   0.08-0.29    0.05-0.11      0.26-0.31
[alpha]-2 globulin, g/dL   0.38-0.80    0.33-0.44      0.75-0.85
[beta] globulin, g/dL      0.21-0.86    0.13-0.29      0.77-0.94
[gamma] globulin, g/dL     0.17-0.44    0.14-0.20      0.40-0.48
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Author:Hausmann, Jennifer C.; Cray, Carolyn; Hartup, Barry K.
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Sep 1, 2015
Words:5286
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