Hematology and plasma chemistry reference values, body mass scores, and fecal

bacteriological analysis in tundra swans, Cygnus columbianus

By

Juliana F. Milani, D.V.M. M.S.

Submitted in Partial Fulfillment of the

Requirements for the Degree

MASTER OF PREVENTIVE VETERINARY MEDICINE

(MPVM)

School of Veterinary Medicine

University of California, Davis

Davis, California

August 2009

Reviewed and Approved by

_________________________________

Rance LeFebvre, Ph.D.

Professor; Veterinary Medicine: Pathology, Microbiology and Immunology

_________________________________

Michael Ziccardi, D.V.M., MPVM, Ph.D.

Assoc. Professor; Veterinary Medicine: Pathology, Microbiology and Immunology

ABSTRACT

Although the tundra swan (Cygnus columbianus) is very numerous and

possesses a wide distribution in North America, it has a very restricted literature. In

order to provide tools for future health-based monitoring programs, we developed

reference intervals for plasma biochemistry, hematology, and body weight (with

comparison among sexes) for 100 adult tundra swans (34 males and 66 females)

sampled in Kotzebue Sound, Alaska, during summer 2008. Moreover, to analyze

possible public health impacts, the prevalence of enteric Salmonella spp. and

Escherichia coli O157:H7 for a subset of 68 swans was attempted. Significant body

condition results included the unsurprising finding that females were significant lighter

than males. For the biochemical analysis, males had higher values in the upper limit of

the calcium interval when compared to females; while females had significant greater

mean levels of potassium. In comparing biochemical ranges to other avian species,

tundra swans were seen to have comparatively high values of creatine kinase, lactate

dehydrogenase, amylase, and alkaline phosphatase, and lower values for cholesterol.

Hematological analyses showed no gender differences, and the comparison with other

avian species demonstrated sizable differences in hematocrit, heterophils, white blood

cell count, and leukocyte differential estimates. There was no growth of either

Salmonella spp. or Escherichia coli O157:H7 in any of the animals sampled. In

conclusion, the fact that tundra swans’ plasma biochemistry and hematology vary

considerably from other avian species reinforces the fact that each avian species must

have its own normal values for ongoing health studies.

INTRODUCTION

Performing comprehensive health assessments on wild bird populations to

determine responses to environmental stressors typically include the evaluation of

physical and biochemical condition, and comparison of these values to established

baseline reference values. Specifically, hematology and plasma/serum biochemistry can

be used to assess the effects of health related problems, such as contaminant

intoxication, malnutrition, and exposure to infection (Sparling et al. 1999), and examine

relationships with important life history parameters (Dunbar et al. 2005). Because of this

association, ranges for biochemical parameters have been reported in the literature for

many common species of waterfowl (Franson et al. 1985, Perry et al. 1986). Body size

and weight are also important measures of individual health, which have been

correlated with both annual survival (Haramis et al. 1986, Conroy et al. 1989, Owen and

Black 1989, Schmutz 1993, Slattery and Alisauskas 2002, Hill et al. 2003) and

reproductive success in migratory waterfowl (Yerkes 2000, Bety et al. 2003). Body

condition in waterbirds can negatively be affected by factors such as population density

(Reed and Plante 1997), lead poisoning (Hohman et al. 1990), and physiologic stress

(Kitaysky et al. 1999). However, due to the broad physiological and seasonal

differences between genders for both biochemical and body measurements, any

baseline reference values must be careful to take this parameter into account,

One migratory bird species where sparse health data exist is the tundra swan

(Cygnus columbianus). Even though this bird is the most numerous and widespread of

the two swan species native to North America (the other being the trumpeter swan,

Cygnus buccinator), literature on this magnificent bird is exceptionally limited. From

information gathered in the last twenty years, only basic aspects related to migration,

breeding and demography are known (Limpert and Earnst 1994); baseline health

assessments and the range of normal blood parameters, with comparison between

genders, have not yet been published.

Another point in favor of better understanding health in tundra swans is related to

their potential role in public health. The tundra swan is a migratory waterfowl that breeds

during the summer in Alaska and Northern Canada on tundra lakes, ponds, and pools.

From there, this species migrates following one of two different routes (Western and

Eastern), wintering in shallow estuaries, lakes, ponds, and rivers on either the Pacific

Coast, Atlantic Coast, or in the interior West and Great Lakes (Limpert and Earnst

1994). Little is known about tundra swans’ potential for harboring and spreading

zoonoses, especially those related to enteric bacteria, and many public health

professionals feel that surveillance programs should include investigation of infectious

zoonotic agents in specific wildlife species (Chomel et al. 2007). In particular, due to

their flight range, migrating birds may easily spread zoonotic enteric bacteria,

contaminating soil, crops, and water. Hagedorn et al. (1999) analyzed the sources of

fecal pollution in a rural Virginia watershed and concluded that about 7% of fecal

streptococci found in the water had come from waterfowl. Furthermore, bacteria that

may be pathogenic to humans and domestic animals, such as Salmonella spp.,

Campylobacter spp., and Yersinia spp., have been previously isolated from seabirds

species (Butterfield et al. 1983, Kapperud and Rosef 1983, Smith et al. 2002).

Therefore, studying tundra swans may be a highly effective way to determine potential

threats in public health.

Beginning in 2006, the tundra swan was identified as a priority species for early

detection of Asian highly pathogenic avian influenza H5N1 (hereafter, Asian HPAI) in

North America (Alaska Interagency HPAI Bird Surveillance Working Group 2006).

Consequently, sampling of swans throughout Alaska significantly increased in 2008 (Ely

et al. 2008, Wilson 2008) though, comprehensive health assessments were sporadically

completed. Therefore, in conjunction with this increased disease detection effort, we

sampled and weighed tundra swans to establish baseline reference values and body

mass measurements for future health-based monitoring programs and examine broad

differences among sexes. In addition, cloacal swabs were collected for a subsample of

swans for isolation of Salmonella spp. and Escherichia coli O157:H7, two of the top five

waterborne zoonotic pathogens of primary human health concern (Craun et al. 2004).

MATERIALS AND METHODS

A population of 100 apparently healthy tundra swans (Cygnus columbianus) – 66

females - were sampled near Kotzebue Sound, Alaska (66°09’N, 162°0’W) during the

summer of 2008, in conjunction with planned Asian HPAI sampling, banding, and

deployment of satellite transmitters. Capture methods for molting swans followed those

outlined by Spindler and Hall (1986). Briefly, individuals from flightless molting flocks

and family groups were captured on foot with dip-nets or from small inflatable boats

deployed by aircraft. Birds were weighed to the nearest 10g using a digital scale, and

up to 6 ml of blood were drawn by jugular or brachial venipuncture from all flightless

birds captured. All sampling was conducted following protocols approved by the U.S.

Geological Survey, Alaska Science Center, Institutional Animal Care and Use

Committee (Assurance No. 2008-06). Immediately following sample collection, 2 mls of

whole blood was transferred to lithium-heparinized Vacutainers® (Becton Dickinson,

Rutherford, New Jersey, USA) and the remaining blood was transferred to sterile serum

separator Vacutainers®. The serum samples were allowed to clot for a minimum of 2

hr, centrifuged at 3000 rpm for up to 10 min, and the serum transferred to 1.5 ml

cryovials (Eppendorf North America, Westbury, New York, USA). Peripheral blood

smears were prepared from whole blood samples for manual differential blood cell

counts. All samples were kept cool (4oC to 8oC) and held no more than 48 hours until

standard avian complete blood count (CBC) and chemistry panels were performed

(Idexx Veterinary Services; Sacramento, CA, USA).

Cloacal samples for the first 68 animals captured were acquired by placing a

sterile BD CultureSwab™ Plus infused with Cary-Blair transport medium (BD

Diagnostics, Franklin Lakes, NJ, USA) into the cloacae and softly revolving the tip

against the mucosal lining. The swabs were transported from Alaska to Davis by plane

at room temperature (15ºC to 25ºC) and subsequently cultured at UC Davis, School of

Veterinary Medicine, Department of Pathology, Microbiology and Immunology. Each

swab was used to inoculate the first quadrant of 5% Sheep Blood Agar, MacConkey

agar, Xylose lysine deoxycholate (XLD) agar (for Salmonella) and MacConkey Sorbitol

(SMAC) agar (for E. coli O157:H7). The plates were then streaked in four quadrants for

isolation and incubated aerobically at 37°C for 24 hours. After incubation, MacConkey

plates were inspected for lactose negative colonies (possible Salmonella); XLD plates

were inspected for hydrogen sulfide positive (H2S+) colonies (Salmonella); and SMAC

plates were inspected for sorbitol negative colonies (E. coli O157:H7). Sheep Blood

Agar plates were used as a backup once swabs were no longer viable after 7 days. Any

suspect pathogens would undergo further biochemical tests towards identification.

Descriptive statistics and histograms for each analyte were calculated using

Minitab® Statistical Software. The reference intervals (including 95% confidence

intervals for lower and upper limits), means, medians, and standard deviations were

calculated for each blood analyte using MedCalc Software (MedCalc Software,

Broekstraat 52, 9030 Mariakerke, Belgium). Variation between sexes was examined

comparing either means (using t-tests, and accepting differences if p-value was smaller

than 0.05) or lower and upper limit confidence intervals - in the case of no overlapping

values, the interval between male and female was considered significantly different (I.

Gardner, pers. comm.).

RESULTS

Body mass

The reference intervals (including 95% confidence intervals (CI) for lower and

upper limits), means, medians, and standard deviations (SD) for animal weight

(categorized by gender) are shown in Table 1. Overall, the weight range went from

5.17Kg to 7.99Kg (median 6.50Kg), and females showed to be significant lighter than

males.

Plasma biochemistry and hematology

The results for plasma biochemical parameters are shown in Tables 2 and 3.

Seven of these 16 reference intervals [albumin globulin ratio (A/G), albumin, amylase

(AMYL), calcium, cholesterol (CHOL), glucose (GLU), and sodium] had 95% CIs

calculated for their upper and lower limits, thus comparisons between male and female

could be made by juxtaposing these bounds (Table 2). The only analyte that showed

significant gender differences was calcium, with males having slightly higher values

than females in the upper limit (the lower limit was considered to be the same).

Regarding the remainder nine parameters [alkaline phosphatase (ALP), aspartate

aminotransferase (AST), creatine kinase (CK), globulin (GLOB), lactate dehydrogenase

(LDH), phosphorus (PHOSP), potassium, total protein (TP) and uric acid (UA)] (Table

3), no 95% CI were able to be calculated due to small sample size in the male group;

therefore assessment of variation between sexes was made by comparison of

parameter’s means, using t-tests. The only analyte that showed gender difference was

potassium, with females having significant higher mean level than males (p=0.0108).

Tables 4 and 5 show the values of hematology results. The 95% CI of the

reference intervals’ bounds were calculable for hematocrit (HCT), heterophils, and

lymphocytes, but no significant difference between males and females were seen

(Table 4). The reference intervals for white blood cells (WBC), basophils, eosinophils,

and monocytes were not able to include 95% CI, and comparison of gender means

showed no significant difference (Table 5).

Fecal Bacteriological Analysis

Regarding the enteric bacteriological analysis, no H2S positive colonies were

found on XLD agar and no sorbitol negative colonies were found on SMAC agar,

meaning that there was no growth of E. coli O157 or of Samonella spp. The majority of

organisms growing on the MacConkey agar plates were lactose positive, indicative of

commensal E. coli.

DISCUSSION

This study establishes baseline reference intervals for blood values and body

weight in wild tundra swans, and examines differences between genders. Moreover,

isolation of enteric E. coli O157:H7 and Salmonella spp. were attempted to determine

zoonotic fecal pathogen prevalence.

Body Mass

The weight intervals of tundra swans evaluated in this study, 5.93Kg – 8.29Kg for

males (mean = 7.11Kg) and 5.10Kg – 7.50Kg for females (mean = 6.30Kg), were in

accordance with that acceptable for the species [adult males: 3.8Kg –10.5Kg (mean =

7.2 kg); and adult females: 4.1Kg – 9.0Kg (mean = 6.3 kg)] (Limpert and Earnst 1994).

Tundra swan females weighed less than males - a similar finding to that in other

commensal species such as free-ranging common loons (Haefele et al. 2005). Body

condition scores were not assessed because more than one person collected

measurements. Since this is a subjective measurement, it can be highly inconsistent

between different veterinarians/biologists, and results may not be directly comparable.

Plasma Biochemistry and Hematology

Our study showed very few differences between male and female tundra swans.

Only plasma calcium and potassium were seen to differ. The small calcium difference

found is expected when blood collection happens soon after egg-laying (Hannon 1979).

A similar result was appreciated in emperor geese (Chen canagica) (Franson et al.

2009). The potassium finding is not well explainable. In spotted dove (Streptopelia

chinesis) and blue rock pigeon (Columbia livia), females had higher potassium levels

than males (Umapathy and Ashokan 1997), but no gender variation was seen in either

king eiders (Somateria spectabilis) (Scott 2006) or captive California condors

(Gymnogyps californianus) (Dujowich et al. 2005). Overall, tundra swans showed

comparably high levels of CK, LDH, amylase and ALP, and low values of cholesterol,

while little to no discrepancy was observed for A/G, albumin, glucose, sodium, AST,

GLOB, PHOSP, potassium, TP and UA, when compared to other avian species such as

emperor geese (Franson et al. 2009), mallards (Anas platyrhynchos) (Fairbrother et al.

1990), king eiders (Scott 2006), captive and wild canvasbacks (Franson et al. 1985,

Perry et al. 1986), barn owls, screech owls, black crowned night herons, and redheads

(Franson et al. 1985) (Tables 2 and 3). According to Williams and Thorne (1996), CK

and LDH can be increased in case of exertional myopathy caused by active or

prolonged captures. The capture method of tundra swans involved the use of light

aircraft to maintain the flock in position for capture, while inflatable boats with two

biologists each captured flightless swans with dipnets, bound their feet with electrical

tape, and transported them to shorelines for processing (Wilson 2008). Many captured

swans were also restrained with custom-made harnesses (Evans and Kear 1972). This

intensive process likely contributed to the release of CK and LDH from muscle tissue

into the blood (Williams and Thorne 1996). Further, avian species at the same

reproductive stage (molting) experiencing muscle-related-stress during capture also

exhibited high values of CK and/or LDH, such as emperor geese (Franson et al. 2009)

and mallards (Fairbrother et al. 1990). The observed changes in ALP, amylase and

cholesterol are of unknown etiologies. In previous studies, female king eiders

demonstrated high levels of ALP and amylase, but blood collection was performed

during breeding and egg-laying phases when both ALP and amylase are expected to

increase due to estrogen-induced hyperproteinemia accompanying egg formation (Scott

2006). Similarly, Fairbrother et al. (1990) found that mallards during egg laying had

higher levels of ALP and amylase than those during molting. However, no similar

comparisons can be made for tundra swans, as there are no published studies

evaluating plasma biochemical results in egg-laying tundra swans. In commercial

turkeys, it has been observed that females carrying a mutating gene that inhibits normal

feather development had significant higher values of ALP and lower values of

cholesterol when compared to females with normal feather development (Zakrzewska

and Savage 1997). One hypothesis for the observed alteration in ALP and cholesterol

encountered in our study could be related to the loss and formation of feather during the

molting stage, but further studies are still needed to better understand the metabolism

involved in this process.

In this study, hematological analyses showed no differences between males and

females; a finding also noted previously in free-ranging common loons (Gavia immer)

(Haefele et al. 2005) and flightless cormorants (Phalacrocorax harrisi) (Travis et al.

2006) (Table 4). Similarly, hematocrit (HTC) values were comparable to those seen in

loons (Haefele et al. 2005) and flightless cormorants (Travis et al. 2006), but were

slightly lower than captive and wild canvasbacks (Perry et al. 1986). Small differences in

HTC may be seen due to dehydration or mild anemia (Haefele et al. 2005). Heterophils

were higher in tundra swans than in common loons (Haefele et al. 2005), king eiders

(Scott 2006) and cormorants (Travis et al. 2006). White blood cell counts (WBC) in our

study were comparable to those in common loons, but higher than king eiders (Scott

2006) and cormorants (Travis et al. 2006). Variation in leukograms in birds may occur

due to subclinical diseases, small traumas, and toxicities, as well as to stress related to

capture and manipulation (Campbell 1997, Fudge 1997). In the leukocyte differential,

tundra swans had similar proportions of basophils, but lower proportions of eosinophils,

lymphocytes and monocytes, as compared to cormorants (Travis et al. 2006); lower

basophils, higher eosinophils and lymphocytes, but similar monocytes than king eiders

(Scott 2006); and lower eosinophils than common loons (Haefele et al. 2005). While

most differences are likely to be species-related, parasitism may explain the

discrepancy encountered in the proportion of eosinophils. Even though eosinophils are

not a reliable measure of parasitism in avian species, parasitized birds may have

eosinophilia (Fudge and Joseph 2000). Further studies comparing level of dehydration

and stress, subclinical diseases, small traumas, and parasitism to hematological values

would be of great contribution to establish baseline normal values in tundra swans.

One weakness of this study lies on the fact that not all parameter gender

comparisons could be made through juxtaposition of their limits, and, consequently, we

had to rely on comparisons of means for some analytes. The vast majority of studies

analyzing blood values show means and standard deviation instead of reference

intervals. Ranges of “normal values” are more appropriate, as they are commonly used

by laboratories, clinicians and veterinarians to compare to patients’ test results. Creating

reference intervals with 95% CI in the lower and upper bounds has the disadvantage

that a large sample and data with appropriate distribution (e.g. normal distribution for

parametric analysis) are needed. According to Lumsden (1998), at least 40 samples

would be necessary in each subgroup analyzed in order to ensure that the 95% CIs for

the limits could be calculated. However, for males our sample was 34. Additional

samples from tundra swans are needed to make more comparable results.

Fecal Bacteriological Analysis

Neither Salmonella spp. nor E. coli O157 was found in any animal sampled. This

is in accordance with preliminary research conducted in California’s central coast that

demonstrated only about 0.5% of wildlife carried enteric E. coli O157 (Department of

Fish and Game 2009). A 1-year study carried out in rehabilitation centers in California

showed that 4% of wildlife were positive for Salmonella spp. (Smith et al. 2002), while

studies in Norway with avian wildlife (Kapperud and Rosef 1983) and in Trinidad with

wild and racing pigeon (Adesiyun et al. 1998) demonstrated that only 0.8% and 1% of

animals yielded Salmonella spp., respectively. One significant difference between these

studies (besides the fact that in California the samples were collected from rehabilitation

centers where animals are weaker and more susceptible to infection) is that feces were

used for the tests in California, while samples were collected with swabs in Trinidad and

Norway. Kotton et al. (2006) demonstrated that the sensitivity of tests using swabs to

detect Salmonella typhimurium in humans was only moderate when compared to fecal

cultures. Thus, the fact that our samples were collected by swabs may have affected

our results by missing potential infected birds. Moreover, it is important to notice that in

order to reach the 4% positive result demonstrated by Smith et al. (2002), we just

needed three positive animals, Therefore, studies using larger sample sizes of fecal

samples are needed to verify whether our results represent accurate prevalence

estimates of these pathogens.

This is the first study to create baseline reference blood values and to assess

possible public health threat of tundra swan. Although the sample size in the male group

was considered small to calculate the 95% CI for the reference intervals bounds, and

the use of swabs may have underestimated the presence of enteric E. coli and

Salmonella spp., the information of this study serves as basis for following researches,

not only in wild tundra swan, but in all wild bird populations. Furthermore, the fact that

values for hematology and plasma biochemistry vary from each avian species indicates

that each species has its own normal values, and emphasizes the importance of studies

evaluating baseline blood analyses in wildlife.

ACKNOWLEDGMENTS

Support for this project was provided by the Calvin Schwabe One Health Project;

the US Fish and Wildlife Service - Alaska Region; and the MPVM Program at the

University of California, Davis.

I would like to thank my advisors, Dr. Michael Ziccardi and Dr. Rance LeFebvre

for their precious guidance during the course of this study and in the writing of this

thesis. In addition, I would like to express my gratitude to Dr. Cheryl Scott, Heather

Wilson, and Dr. Ian Gardner for their support and expertise during planning,

implementation, and data analyzing of this experiment, and to the veterinarian Dr. Scott

Ford, for the training and help. I would also like to thank the Alaskan USFWS biologists

and personnel Brad Scotton, Karen Bollinger, Bill Larned, Paul Anderson, Nate Olson,

Rob MacDonald, Dennis Marks, Rachael Schively and Tina Moran, and the UC Davis

Microbiology Laboratory staff research associates Poyin Chen and Heather A. Van

Hoosear for their planning, implementation, expert assistance, and invaluable support.

Finally, I would like to express my appreciation to everyone that, by one way or

another, had any participation on this project, directly or indirectly, but whose names

would be impossible to mention in only one page.

Table 1. Reference intervals (including 95% confidence intervals (CI) for lower and upper limits), means, medians, and standard deviations (SD) of tundra swans’ weight (in Kg), sampled at Kotzebue Sound, Alaska, in summer 2008.

Reference Intervals

Lower Upper

Limit 95% CI Median Limit 95% CI Mean SD

Total 5.17 4.97 - 5.38 6.50 7.99 7.78 - 8.19 6.58 0.72

Male 5.93 5.64 - 6.23 7.16 8.29 8.00 - 8.59 7.11 0.60

Female 5.10 4.89 - 5.32 6.32 7.50 7.29 - 7.72 6.30 0.61

Total n=100, Female n=66, Male n=34 (n=number of animals sampled)

Table 2. Reference intervals (including 95% confidence intervals (CI) for lower and upper bounds), means, medians, and standard deviations (SD) for 7 of the 16 biochemical parameters of tundra swans, sampled at Kotzebue Sound, Alaska, in summer 2008.

Lower Upper

Limit 95% CI Median Limit 95% CI Mean SD

A/G Total 0.44 0.42 - 0.46 0.60 0.72 0.7 - 0.74 0.58 0.07

Male 0.45 0.41 - 0.48 0.60 0.72 0.69 - 0.76 0.59 0.07

Female 0.43 0.41 - 0.46 0.60 0.72 0.7 - 0.75 0.58 0.07

Albumin Total 1.05 1.01 - 1.08 1.30 1.52 1.49 - 1.56 1.28 1.12

g/dL Male 1.07 1.02 - 1.13 1.30 1.54 1.48 - 1.6 1.31 0.12

Female 1.03 0.99 - 1.08 1.30 1.51 1.47 - 1.56 1.27 0.12

AMYL Total 2246.00 2246 - 2246 4031.50 6247.00 6247 - 6247 4133.00 1140.22

(U/L) Male 2261.30 2261.3 - 2261.3 3831.00 6185.40 6185.4 - 6185.4 3889.62 1102.24

Female 2329.70 2329.7 - 2329.7 4072.00 7331.35 7331.35 - 7331.35 4258.38 1147.34

Calcium* Total 8.28 8.18 - 8.38 9.00 9.70 9.59 - 9.8 8.99 0.36

mg/dL Male 8.35 8.16 - 8.54 9.15 9.88 9.69 - 10.07 9.11 0.39

Female 8.28 8.16 - 8.39 9.00 9.57 9.45 - 9.68 8.92 0.33

CHOL Total 76.89 71.82 - 81.97 108.50 146.55 141.47 - 151.62 111.72 17.77

mg/dL Male 81.02 72.22 - 89.82 113.00 150.92 142.12 - 159.72 115.97 17.83

Female 75.30 69.14 - 81.45 106.50 143.76 137.61 - 149.92 109.53 17.47

GLU Total 201.57 188.2 - 214.94 289.50 384.95 371.58 - 398.32 293.26 46.78

mg/dL Male 195.47 170.03 - 220.91 289.00 397.53 372.09 - 422.97 296.50 51.55

Female 204.47 188.8 - 220.14 289.50 378.71 363.05 - 394.38 291.59 44.45

Sodium Total 144.37 143.79 - 144.96 148.00 152.43 151.84 - 153.01 148.40 2.05

mEq/L Male 144.72 143.67 - 145.78 149.00 153.10 152.05 - 154.16 148.91 2.14

Female 144.26 143.57 - 144.96 148.00 152.01 151.31 - 152.71 148.14 1.98

Total n=100, Female n=66, Male n=34 (n=number of animals sampled) * Male values in the upper bound were significantly higher than those of females

Table 3. Reference intervals, means, medians, and standard deviations (SD) for 9 of the 16 biochemical parameters of tundra swans, sampled at Kotzebue Sound, Alaska, in summer 2008, where 95% CIs could not be calculated.

Lower Upper

Limit Median Limit Mean SD

ALP Total 169 677.00 1544.00 720.88 334.43

(U/L) Male 276.75 683.00 1248.45 706.15 230.97

Female 90.8 672.50 1617.1 728.47 378.27

AST (U/L) Total 12 26.00 162.00 35.01 33.82

Male 11.35 24.50 111.20 31.00 23.03

Female 12 26.00 172.20 37.08 38.21

CK Total 260 957.00 6936.00 1576.72 2310.47

(U/L) Male 242.55 801.00 6054.70 1190.85 1288.11

Female 271.55 984.00 8363.25 1775.50 2677.73

GLOB Total 1.80 2.20 3.00 2.23 0.31

g/dL Male 1.70 2.25 3.10 2.26 0.35

Female 1.80 2.20 2.99 2.22 0.29

LDH Total 312.00 652.00 1365.00 670.50 273.38

(U/L) Male 335.80 644.50 1049.05 636.59 214.97

Female 305.20 660.00 1565.60 687.97 299.09

PHOSP Total 0.5 2.35 5.40 2.42 1.43

mg/dL Male 0.5 2.20 5.46 2.34 1.45

Female 0.62 2.45 5.27 2.45 1.42

Potassium* Total 2.00 2.50 3.70 2.60 1.26

mEq/L Male 1.97 2.45 3.03 2.48 0.26

Female 2.00 2.60 3.87 2.66 0.43

TP Total 2.90 3.45 4.60 3.52 0.40

g/dL Male 2.84 3.55 4.50 3.57 0.44

Female 3.00 3.40 4.56 3.49 0.38

UA Total 4.40 7.30 13.50 7.87 2.62

mg/dL Male 4.88 7.95 18.46 8.34 3.02

Female 4.32 7.20 12.84 7.63 2.38

Total n=100, Female n=66, Male n=34 (n=number of animals sampled) * Male mean values were significantly lower than those of females.

Table 4. Reference intervals (including 95% confidence intervals (CI) for lower and

upper bounds), means, medians, and standard deviations (SD) for 3 of 7

hematologic parameters of tundra swans, sampled at Kotzebue Sound, Alaska,

in summer 2008.

Lower Upper

Limit 95% CI Median Limit 95% CI Mean SD

HCT Total 39.02 38.18 - 39.85 45.00 50.49 49.66 - 51.33 44.76 2.93

(%) Male 39.58 38.21 - 40.95 45.00 50.45 49.08 - 51.82 45.01 2.77

Female 38.71 37.65 - 39.77 45.00 50.53 49.47 - 51.59 44.62 3.02

Heterophils Total 44.29 40.96 - 47.62 68.50 89.91 86.58 - 93.24 67.10 11.64

(%) Male 40.89 34.22 - 47.56 66.50 93.87 87.2 - 100.54 67.38 13.52

Female 46.07 42.32 - 49.83 69.00 87.84 84.08 - 91.59 66.95 10.65

Lymphocytes Total 9.34 6.66 - 12.02 27.00 45.93 43.25 - 48.61 27.64 9.33

(%) Male 6.85 1.6 - 12.1 28.00 47.94 42.69 - 53.19 27.39 10.48

Female 10.53 7.44 - 13.63 26.50 44.98 41.88 - 48.08 27.76 8.79 Total n=100, Female n=66, Male n=34 (n=number of animals sampled)

Table 5. Reference intervals, means, medians, and standard deviations (SD) for 4 of 7

hematologic parameters of tundra swans, sampled at Kotzebue Sound, Alaska,

in summer 2008, where 95% CIs could not be calculated.

Lower Upper

Limit Median Limit Mean SD

WBC Total 7.30 15.90 40.00 19.21 9.37

(x1000) Male 6.72 15.15 47.35 18.26 10.33

Female 7.30 17.25 39.81 19.70 8.88

Basophils Total 0 1 4 1.29 1.12

(%) Male 0 1 3.65 1.09 1.00

Female 0 1 4 1.39 1.18

Eosinophils Total 0 2 13 3.30 4.02

(%) Male 0 1.5 13 2.94 3.49

Female 0 2 16.4 3.48 4.28

Monocytes Total 0 0 3 0.37 0.85

(%) Male 0 0 1.65 0.21 0.48

Female 0 0 3.85 0.45 0.98 Total n=100, Female n=66, Male n=34 (n=number of animals sampled)

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