SERUM PROTEIN CAPILLARY ELECTROPHORESIS AND MEASUREMENT OF ACUTE PHASE PROTEINS IN A CAPTIVE CHEETAH (ACINONYX JUBATUS) POPULATION Author(s): Sarah Depauw, D.V.M., Ph.D., Joris Delanghe, D.M.Sc., Ph.D., Katherine Whitehouse-Tedd, Ph.D., Mads Kjelgaard-Hansen, D.V.M., Ph.D., Michelle Christensen, D.V.M., Ph.D., Myriam Hesta, D.V.M., Ph.D., Dipl. E.S.V.C.N., Pierrot Tugirimana, D.M.Sc., Ph.D., Jane Budd, B.V.M.S., Veronique Dermauw, D.V.M., Ph.D., and Geert P. J. Janssens, D. Eng., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 45(3):497-506. 2014. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0111R1.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0111R1.1

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Journal of Zoo and Wildlife Medicine 45(3): 497–506, 2014 Copyright 2014 by American Association of Zoo Veterinarians

SERUM PROTEIN CAPILLARY ELECTROPHORESIS AND MEASUREMENT OF ACUTE PHASE PROTEINS IN A CAPTIVE CHEETAH (ACINONYX JUBATUS) POPULATION Sarah Depauw, D.V.M., Ph.D., Joris Delanghe, D.M.Sc., Ph.D., Katherine Whitehouse-Tedd, Ph.D., Mads Kjelgaard-Hansen, D.V.M., Ph.D., Michelle Christensen, D.V.M., Ph.D., Myriam Hesta, D.V.M., Ph.D., Dipl. E.S.V.C.N., Pierrot Tugirimana, D.M.Sc., Ph.D., Jane Budd, B.V.M.S., Veronique Dermauw, D.V.M., Ph.D., and Geert P. J. Janssens, D. Eng., Ph.D.

Abstract: Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non–species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P , 0.001) serum amyloid A, a2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P ¼ 0.020), a2-globulin (P , 0.001) and haptoglobin (P ¼ 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non– species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease. Key words: Amyloid, felids, globulins, haptoglobin, inflammation.

INTRODUCTION Morbidity in the captive cheetah (Acinonyx jubatus) population continues to cause concern. Although these animals are classified as vulnerable on the IUCN Red List, captive breeding results are rather poor and renal and gastrointestinal diseases are highly prevalent.17,22,23,30 From the Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B9820 Merelbeke, Belgium (Depauw, Hesta, Dermauw, Janssens); Department of Clinical Chemistry, Ghent University Hospital, 9000 Ghent, Belgium (Delanghe, Tugirimana); Cheetah Outreach, 209–211 Victoria Junction, Prestwich Street, Cape Town 7700–8099, Western Cape, South Africa (Whitehouse-Tedd); Central Laboratory, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, 2000 Frederiksberg, Denmark (Kjelgaard-Hansen, Christensen); Breeding Centre for Endangered Arabian Wildlife, P.O. Box 29922, Sharjah, United Arab Emirates (Budd, Whitehouse-Tedd); and School of Animal Rural and Environmental Sciences, Nottingham Trent University, Southwell, NG25, OQF, United Kingdom (Whitehouse-Tedd). Correspondence should be directed to Dr. Depauw (sarahdepauw3@gmail. com).

Currently, health monitoring of captive cheetahs is mainly based on the evaluation of clinical signs and the interpretation of hematology and blood biochemistry results. Consequently, illness in cheetahs is often only diagnosed at a late stage of disease, when clinical signs are severe or general health parameters are abnormal. As such, zoo veterinarians and researchers are often hampered in their ability to define the etiology of common diseases and to increase their prevention or successful treatment in this captive population. Serum proteins are an important group of molecules essential to normal animal physiology, and the fluctuation of a single serum protein can be altered by several factors, such as immunodeficiency, as well as infectious and parasitic disease or malignancy.27 Electrophoresis enables the separation of serum proteins into six fractions (albumin and a1-, a2-, b1-, b2-, and c-globulins), resulting in a typical electrophoretic pattern for the distribution of proteins. Alterations in this pattern are recognized as a useful tool in the diagnosis, prognosis, and monitoring of various

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diseases in both human and veterinary medicine.7,27,32,35 Globulin fractions in the electrophoretogram also make up acute-phase proteins (APP), such as a1-acid glycoprotein, a1-antitrypsin, haptoglobin (Hp), ceruloplasmin, C-reactive protein (CRP), the C3 portion of complement, and fibrinogen, which can increase in response to acute inflammation, malignancy, or trauma. APP rapidly increase when inflammation occurs and rapidly decrease after elimination of the inflammation.11 These characteristics make them highly sensitive biomarkers for the early diagnosis of inflammatory conditions and progress evaluation. In comparison with leukocyte counts, APP exhibit a faster response time and have a longer analytical stability, which explains their high sensitivity.33 Until now, no information was found on serum protein electrophoresis and APP concentrations during health and inflammation in any exotic feline species. Although currently several commercially produced assays are available for the determination of APP in dogs9,16,18,20,24 and cats,2,12 these immunologic assays are often expensive and do not always guarantee sufficient antigen crossreactivity between species.3 Moreover, antibodies against specific serum proteins of cheetahs, or any other exotic felid, are not commercially available. Consequently, there is a high need for alternative tests to measure APP in these species. The current study presents capillary electrophoresis of serum proteins in 80 captive cheetahs from diverse geographic regions, with varying husbandry and medical backgrounds. In addition, methods for measuring the concentrations of APP serum amyloid A (SAA), CRP, and Hp were assessed in the captive cheetah.

MATERIALS AND METHODS Sera Sera were opportunistically collected from zoologic institutions in Europe and the United Arab Emirates. A request for donation of cheetah blood samples was drafted and approved by the European Endangered Cheetah Program, within the European Association of Zoos and Aquaria (EAZA), and circulated among EAZA members. In total, nine zoologic institutions responded to the request and 80 serum samples were collected from 61 individual animals. Age, sex, subspecies (Acinonyx jubatus soemmeringii, A. j. jubatus), reason for blood draw and date of sampling were recorded for all samples. Detailed information on the diet, medical background, and health status

was provided by the respective zoo veterinarians. Hematology or serum biochemistry results were not provided. Blood samples were centrifuged at 1,000 3 g for 10 min, and sera were stored at 208C until further transport on dry ice to the Laboratory of Animal Nutrition, Ghent University, where they were stored at 808C for a maximum of 18 mo. Repetitive samples from the same individuals with more than 1 yr between sampling were viewed to be independent. The collection represented sera from 33 female and 47 male captive cheetahs with a median age of 7.9 yr (range: 0.5– 14.5 yr), with samples from 43 A. j. soemmeringii and 37 A. j. jubatus. Based on information provided for medical background, cheetahs were categorized as healthy (n ¼ 47), unhealthy (n ¼ 14), suspected unhealthy (n ¼ 14), or unknown (n ¼ 5). Out of 80 samples, 38 were obtained for independent ongoing research projects; 4 for transport certification, 9 for chronic kidney failure, 5 for gastrointestinal problems, 13 because of suspicion of an infectious disease, 6 for other noninfectious medical problems, and 5 for unknown reasons. Within this studied cheetah population, 63% were fed a mixture of meat-only and wholecarcass diets, 10% meat only, 18% whole carcass only; 9% did not have diet reported. Serum protein capillary electrophoresis Aliquots of 100 ll cheetah serum were thawed at room temperature and vortexed. Serum proteins were characterized by capillary electrophoresis (The Capillarys Protein(e) 6 kit (PN 2003) in combination with the CapillarysTM 2 CE system, ´ vry 91000, France), which allowed the Sebia, E separation of proteins into six fractions (c-, b1-, b2-, a1-, and a2-globulins and albumin). Prior to hydrodynamic injection (4s), 40 ll of serum was automatically diluted five times in the running buffer (pH 9.9). The proteins were separated in eight fused-silica capillaries (effective length 15.5 cm, internal diameter 25 lm; optical cell 100 lm), applying 7kV for 4 min at 35.58C (Peltier device) and proteins were directly detected by their absorbance at 200 nm (deuterium lamp). Total protein concentrations were measured spectrophotometrically (Cobas 8000 Analyzer, Roche Diagnostics, Basel 4070, Switzerland). To obtain serum protein concentrations, the percentage volume of each fraction was multiplied by the total protein concentration of the representative sample.

DEPAUW ET AL.—SERUM PROTEIN ANALYSES IN THE CHEETAH

Determination of Hp concentrations Determination of the a2-sialoprotein Hp was based on its hemoglobin (Hgb)-binding capacity.25 Because Hp-bound Hgb shows a significantly changed protein spectrum during electrophoresis, comparison of absorbance values between free Hp and Hp-Hgb complex enables the identification of Hp within the a2fraction.34 To identify the absorbance of Hp within the a2-fraction, eight serum samples were selected from the collection. Four samples represented low a2-concentrations, and another four high a2-concentrations. In these two groups, both subspecies were equally present. To make Hgb lysate (0.1 g Hgb/dl), red blood cells (1 ml EDTA-blood, human) were washed three times with 9 ml saline. Thereafter, 2 ml of distilled water and 0.5 ml of saline was added to enable hemolysis. The samples were centrifuged (2,000 3 g) for 2 min, and the supernatant was extracted and stored at 208C. Aliquots of 100 ll cheetah serum were thawed at room temperature and vortexed. In each sample, 100 ll of Hgb lysate was added. The serum samples with Hgb lysate were incubated for 15 min at 378C before the a2fraction was reanalyzed by capillary electrophoresis. The exact location of the serum Hp peak, within the a2-fraction, was determined by visual inspection of the electrophoretogram peaks of free Hp versus Hp-Hgb complex of these eight samples. After Hgb supplementation of the serum, the initial Hp peak disappeared on the electrophoretogram and was integrated in a newly formed peak composed of Hgb-Hp complexes. After confirmation of the Hp peak location, the area under the curve of the Hp peak was determined for all serum samples. To obtain Hp concentrations, the percentage volume of Hp was multiplied by the total protein concentration of the representative sample. Determination of SAA concentrations Serum SAA concentrations were measured via a recently developed latex agglutination turbidimetric immunoassay (LAT; Eiken Chemical Co., Bunkyo-ku, Tokyo 113-8408, Japan) using an automated analyzer (ADVIA 1800 Chemistry System, Siemens Healthcare Diagnostics Inc., Tarrytown, NY 10591, USA). This immunoassay has previously been validated for diagnostic measurements of SAA in dogs, horses, and domestic cats.6 All samples were analyzed in duplicate. Results are expressed as mg/L of

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human SAA equivalents because of the use of human calibration material. To confirm sufficient cross-reactivity, the specificity of the monoclonal anti-human SAA antibodies used in LAT for the detection of SAA in captive cheetahs was assessed by Western blot analysis. In particular, it was assessed whether the antibody target;s molecular weight corresponded to the expected molecular weight of SAA. Therefore, serum samples from two captive cheetahs with high concentrations of SAA (assessed to be 845 mg/L and 1,122 mg/l, respectively, using the LAT), as well as one serum sample with undetectable concentration of SAA (negative control), were used in the investigation of the specificity of antibodies in the present study. Samples were diluted 1:50 in trypsin-buffered saline (TBS) and denatured by dilution in a sample reducing agent (NuPAGE Sample Reducing Agent, Life Technologies, Naerum DK-2850, Denmark) and buffer (NuPAGE Sample Buffer, Life Technologies, Naerum DK-2850, Denmark) prior to 5 min of boiling. After application of 15 ll of each denatured sample and 10 ll prestained standard (Seablue Prestained Standard Invitrogen, Life Technologies, Naerum DK-2850, Denmark) on a precast polyacrylamide gel (NuPAGE Novex Bis-Tris MiniGel (12% acrylamide) Invitrogen, Life Technologies, Naerum DK-2850, Denmark), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed at 200 V for 1 hr using an SDS running buffer (NuPAGE MES SDS Running Buffer Invitrogen, Life Technologies, Naerum DK2850, Denmark). Electroblotting was performed onto a transfer membrane (Immobilon-P PVDF Transfer Membrane, Merck Millipore, Darmstadt 64295, Germany) at 150 mA for 1 hr prior to blocking for 10 min using 2% Tween in TBS. After separation of the lane containing the prestained standard (Eiken Chemical Co., Bunkyo-ku, Tokyo 113-8408, Japan), the membrane was washed 336 min in 0.1% Tween in TBS and incubated in monoclonal anti-human SAA antibodies (SAA-1, Eiken Chemical Co., Bunkyo-ku, Tokyo 113-8408, Japan) for 1 hr after 1:1,000 dilution in 0.1% Tween in TBS. After an additional washing step of 336 min in 0.1% Tween in TBS, the membrane was incubated in alcalic phosphatase conjugated goat anti-rat IgG (Roche Applied Science, Penzberg 82377, Germany) for 1 hr after a 1:2,000 dilution in 0.1% Tween in TBS. After a washing step of 3 3 6 min in 0.1% Tween in TBS, the membranes were incubated in 5-bromo-4-chloro3-indonyl phosphate toludine and nitro blue

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tetrazolium chloride (Roche Applied Science, Penzberg 82377, Germany) until visualization of bands. Determination of CRP concentrations Serum CRP concentrations were determined using a species-nonspecific functional CRP assay based on phosphocholine interaction adapted from Tugirimana et al.37 Serum samples were thawed and vortexed, and 10 ll of serum was diluted in 960 ll of Tris-calcium chloride buffer (0.1 mol/L Tris, 0.1 mol/L calcium chloride, with pH adjusted until 7.5 by hydrochloric acid (1 mol/ L)). In addition, 20 ll of a fat emulsion for intravenous infusion (Structokabiven, Fresenius Kabi AB, Upsala 75309, Sweden) was added to the diluted sample. The prepared samples were incubated (378C) for 30 min, followed by intensive shaking. The CRP–phospholipid complexes were turbidimetrically and bichromatically (660 nm/ 700 nm) quantified on a chemistry analyzer (Cobas 6000 Analyzer, Roche Diagnostics, Basel 4070, Switzerland) by measuring lipemic index, as described by Tugirimana et al.37 Statistical analyses Reference intervals were evaluated following the ‘‘Guidelines for the Determination of Reference Intervals in Veterinary Species and other related topics.’’10 Distribution analysis and descriptive statistics were examined with Reference Value Advisor v2.1 (National Veterinary School, Toulouse, France), which is a set of macroinstructions for Excel (Microsoft Office 2010, Microsoft Corporation, Redmond, Washington 98052, USA) that compute reference intervals using the standard and robust methods with and without generalized Box-Cox data transformation. Distribution analysis was performed using the Anderson-Darling statistic. All data had a non-normal distribution. Upper and lower limits of reference intervals were computed by robust methods for transformed and untransformed data, and a 90% confidence interval was assessed for each upper and lower reference value. No data were excluded from the dataset. Effects of health status and disease type on serum protein electrophoresis and APP were tested using the Kruskal-Wallis Test (Post Hoc: Dunn’s test) for data with non-normal distribution using SPSS version 20 (SPSS Inc., Chicago, IL 60606-6412, USA). Effects of gender, age, subspecies, and diet on serum protein electrophoresis and APP were tested in cheetahs categorized

Figure 1. Representative electrophoretogram of cheetah serum, illustrating the separation of proteins into different fractions.

as healthy using the Kruskal-Wallis Test (post hoc: Dunn test). Differences with a P value less than 0.05 were considered significant.

RESULTS Serum protein electrophoresis and Hp determination Capillary electrophoresis successfully separated cheetah serum proteins into albumin and the a1-, a2-, b1-, b2-, and c-globulin fractions, consistent with serum protein fractions of domestic cats (Fig. 1).35 All eight cheetah sera with added Hgb lysate showed a similar marked change in protein spectrum within the a2-fraction. An increase in a2globulins was demonstrated in one individual (Fig. 2a); adding Hgb lysate yielded an altered absorbance and decrease of the a2-peak in this animal (Fig. 2b), confirming that this peak reflects the protein Hp. Albumin and globulin concentrations of cheetahs categorized as healthy fell within reference intervals for domestic cats, with the exception of higher concentrations of b1-globulin in healthy cheetahs compared with domestic cats (Table 1). The concentrations of a2-globulin and Hp were higher (P , 0.001) in unhealthy cheetahs compared with the healthy animals (Table 2). Consequently, the calculated ratio of albumin to globulins was significantly lower in unhealthy cheetahs (P , 0.001). In addition, Hp concentrations in unhealthy cheetahs were higher (P ¼ 0.012) than in cheetahs categorized as suspected unhealthy (Table 2). Moreover, cheetahs with chronic kidney failure (n ¼ 9) had higher (P ¼ 0.001) a2-globulin concentrations (median 10.5 g/ L (min 3.84, max 19.4)) compared with cheetahs from the research group (n ¼ 38; median 3.93 g/L (min 2.19, max 7.80)). Likewise, cheetahs with

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DEPAUW ET AL.—SERUM PROTEIN ANALYSES IN THE CHEETAH

Figure 2. (a) Electrophoretogram from a cheetah with increased a2-globulins.

chronic kidney failure showed higher (P , 0.001) Hp concentrations (median 8.76 g/L (min 2.86, max 15.3)) compared with the research group (median 2.97 g/L (min 1.46, max 5.05)). Neither gender nor diet influenced the outcome of albumin, globulin, and Hp concentrations. In contrast, c-globulin concentrations were higher (P ¼ 0.014) in healthy cheetahs between 8 and 11 yr of age (n ¼ 13, median 7.84 g/L (min 5.43, max 10.8)) compared with healthy cheetahs between 2 and 5 yr of age (n ¼ 4, median 4.32 g/L (min 2.88, max 5.44)). Subspecies also affected serum protein concentrations in healthy animals. A. j. jubatus (n ¼ 16) had higher (P ¼ 0.001) a1-globulin concentrations (median 5.28 (min 3.79, max 8.14) compared with A. j. soemmeringii (n ¼ 30) (median 4.10 g/L (min 2.64, max 6.34)). A lower (P ¼ 0.002) c-globulin concentrations was observed in

Figure 2. (b) Electrophoretogram from the same cheetah after supplementing the serum sample with hemoglobin lysate (final concentration: 0.1 Hgb g/dl), illustrating the altered spectral absorption of haptoglobin (Hp) due to its Hgb-binding capacity. The a2-globulin fraction is markedly increased due to the formation of the Hp-Hgb complexes. The presence of a small excess amount of free Hgb explains the change in the b1-region.

the cheetahs from the A. j. jubatus subgroup (median 5.43 g/L (min 2.88, max 9.56) versus 7.20 g/L (min 4.33, max 20.3)). SAA determination Western blot analyses revealed distinct bands around 10–12 kDa detected in serum from two captive cheetahs (Fig. 3, lanes 1,2). As expected, no bands were detected in the negative control (lane 3). The molecular weight of SAA has previously been shown to be 10–15 kDa in several

Table 1. Serum protein electrophoresis and acute-phase proteins in healthy captive cheetahs opportunistically sampled from multiple institutions.a

Unit

Medb

Min

Max

RI

Albumin g/L a1-globulin g/L a2-globulin g/L b1-globulin g/L b2-globulin g/L c-globulin g/L A:G SAA mg/L

47 41.9 47 4.51 47 4.33 47 4.51 47 3.15 47 6.62 47 1.65 47 31.4

30.3 2.64 2.19 8.82 1.58 2.88 1.07 22.8

55.3 8.14 7.29 9.41 8.69 20.3 2.17 78.1

34.4–51.0 2.55–7.44 2.20–7.45 4.51–8.82 1.87–5.70 3.50–13.6 1.16–2.17 23.6–57.0

Hp

47

CRP

g/L

n

3.13b 1.46 6.81 1.61–6.31

mg/L 47 26.7

0.64 69.5 3.91–58.5

Lower ref lim Upper ref lim (90% CI) (90% CI)

32.8; 2.30; 1.89; 4.02; 1.66; 2.97; 1.08; 22.7;

36.3 2.89 2.49 4.99 2.16 4.08 1.25 25.1

48.1; 6.71; 6.61; 8.32; 4.89; 11.0; 2.05; 46.9;

53.7 8.21 8.27 9.30 6.69 17.5 2.28 76.4

1.41; 1.84

5.43; 7.33

0.89; 8.53

49.9; 65.2

Ref values (domestic cats)

29.0–46.7 2.08–4.99 2.94–10.3 1.54–4.50 1.51–4.90 4.33–21.4

Taylor Taylor Taylor Taylor Taylor Taylor

et et et et et et

al., al., al., al., al., al.,

2010 2010 2010 2010 2010 2010

29.1–44.7 Kajikawa et al., 1999 1.60–3.59 Kajikawa et al., 1999

Med indicates median; Min, minimum; Max, maximum; RI, reference interval; Ref lim, reference limit; CI, confidence interval; A:G, albumin-globulin ratio; SAA, serum amyloid A; Hp, haptoglobin; CRP, C-reactive protein. b Median, RI, and 90% CI for the upper and lower reference limits were determined by the central 95% robust method with and without generalized Box-Cox data transformation. a

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Table 2. Comparison of serum protein electrophoresis and acute-phase proteins in captive cheetahs categorized as healthy, unhealthy, or suspected unhealthy.a Healthy cheetahs (n ¼ 47b)

Albumin (g/L) a1-globulin (g/L) a2-globulin (g/L) b1-globulin (g/L) b2-globulin(g/L) c-globulin (g/L) A:G SAA (mg/L) Hp (g/L) CRP (mg/L)

Unhealthy cheetahs (n ¼ 14c)

Suspected unhealthy cheetahs (n ¼ 14)

Med

Min

Max

Med

Min

Max

Med

Min

Max

41.9 4.51 4.33e 6.67 3.15 6.62 1.65e 31.4e 3.13e 26.7

30.3 2.64 2.19 4.06 1.58 2.88 1.07 22.8 1.46 0.64

55.3 8.14 7.29 9.41 8.69 20.3 2.17 78.1 6.81 69.5

37.6 3.95 9.46f 7.35 3.80 7.67 1.26f 58.1f 8.00f 32.0

33.5 1.65 2.85 3.81 2.32 3.02 0.67 35.7 2.56 7.3

53.3 7.16 19.4 12.1 11.9 14.9 1.99 806 15.3 78.4

41.4 4.16 5.62e,f 7.66 3.09 6.60 1.61e,f 35.1e,f 3.37e 25.6

32.6 2.79 2.14 3.43 2.08 2.66 0.91 23.2 1.60 12.5

66.3 7.53 15.9 13.1 5.33 25.0 2.04 1,122 14.3 39.1

d

a Median indicates median; Min, minimum; Max, maximum; A:G, albumin-globulin ratio; SAA, serum amyloid A; Hp, haptoglobin; CRP, C-reactive protein. b One repetitive sample from six individuals and three repetitive samples from two individuals, .1 yr between sampling. c One repetitive sample from two individuals, .1 yr between sampling. d Medians of the measured concentrations were determined by the central 95% robust method with and without generalized Box-Cox data transformation. e,f Means in the same line with different superscripts are significantly different (P , 0.05).

other species,13,31 and therefore the bands found in the present study are compatible with SAA. In cheetahs categorized as healthy, the SAA reference interval was comparable with reference ranges for domestic cats (Table 1). SAA concentrations were higher (P , 0.001) in cheetahs categorized as unhealthy compared with the healthy individuals (Table 2). In addition, cheetahs with chronic kidney failure (n ¼ 9) had higher (P ¼ 0.020) SAA concentrations (median 39.0 mg/ L (min 36.4, max 391)) compared with cheetahs from the research group (n ¼ 38; median 30.4 mg/ L (min 22.8, max 78.1)). In cheetahs that were suspected to be unhealthy, maximum concentrations of 1,122 mg SAA/L were detected. In the healthy cheetah population, sex affected SAA concentrations; male cheetahs (n ¼ 28) had higher (P ¼ 0.018) SAA concentrations compared with female cheetahs (n ¼ 19; median: 29.8 (min 31.4, max 40.8) versus median 33.0 (min 22.6, max 50.2)). CRP determination Serum CRP concentrations in captive cheetah categorized as healthy are shown in Table 1. No significant effects of gender, age, subspecies, or diet on CRP concentrations in cheetah serum were present in this study. Also, the health status or reason for blood draw did not significantly affect CRP concentrations (Table 2). Levels as high as 78.4 mg CRP/L were detected in one cheetah suffering from chronic kidney disease.

DISCUSSION This study explored the use of capillary electrophoresis, immunoassays, and non–species-specific functional tests to evaluate serum globulin and APP concentrations in captive cheetahs with varying health status. In general, serum capillary electrophoresis of cheetah serum resulted in an electrophoretogram comparable with that of domestic cats,35 although obtained by a different electrophoresis method. The electrophoretogram separated the proteins into albumin and a1-, a2-, b1-, b2-, and c- globulins, as in other mammals. In other species, the a1-globulin fraction mainly consists of the APP a1-acid glycoprotein (AGP), but other proteins, such as a1-antitrypsin and lipoproteins, are also within the constituents of the a1-globulin fraction.27 In domestic cats, AGP is a major APP, meaning that most often this protein will increase during acute inflammation.5 In the studied healthy captive cheetah population, the a1-globulin fraction was comparable with reference ranges found in the domestic cat, and no distinct differences were observed based on health status of the cheetahs. Therefore, it is uncertain whether AGP might also be a major APP in the cheetah. Of interest were the significantly lower a1-globulin concentrations in A. j. soemmeringii compared with A. j. jubatus. Subspecies clinical differences have not been reported in other feline species and warrant further investigation in cheetahs, although it cannot be excluded that this difference was confounded with other

DEPAUW ET AL.—SERUM PROTEIN ANALYSES IN THE CHEETAH

Figure 3. Immunostained Western blot after sodium dodecyl sulfate–polyacrylamide gel electrophoresis of serum from two captive cheetahs with high levels of serum amyloid A (SAA) (detected with latex agglutination turbidimetric immunoassay (LAT)). The monoclonal anti-human SAA antibody used in the LAT was used for immunodetection. Distinct bands with a molecular weight around 10–12 kDa were detected in each of the serum samples (lanes 1,2), whereas no SAA was measured in a negative control of cheetah serum (lane 3). M: prestained standard (Seablue Prestained Standard Invitrogen, Life Technologies, Naerum DK2850, Denmark).

factors inherent to differences in management between both subspecies. The a2-globulin fraction consists principally of the APP a2-macroglobulin and Hp, but other proteins, such as ceruloplasmin, also migrate there.27 Hp has a very high affinity for binding Hgb and functions primarily to prevent iron loss and kidney damage during hemolysis.21 This specific characteristic of Hp provides the opportunity to explore its use as a functional test to determine Hp concentrations instead of screening immunologic tests, which require specific antigen reactivity. Until now, Hp has not been determined using an electrophoresis-based methodology in any felid. However, in the current study, the behavior of Hp-bound Hgb on electrophoresis of cheetah sera was similar to the one previously observed in human sera using spectrophotometry.34 As in human beings, complexation of Hgb to

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Hp showed an alteration of the Hp band on electrophoresis, which allowed the identification of the Hp peak within the a2 fraction. It is likely that the mobility of Hp-bound Hgb within the a2 area is comparable for all mammalian species, which makes this functional test a useful tool for other exotic felids, as well as a broader range of species. In the current study, whole blood was not available and therefore Hgb lysate was made from human blood. It is unlikely that this affected the Hp analysis. However, in future studies it is recommend to use Hgb lysate from the studied animal where possible. Cheetahs categorized as healthy had comparable a2-globulin and Hp concentrations as found in healthy domestic cats, although the upper Hp reference limit was higher in cheetahs.14,35 Although the medical background of the sampled animals was screened, details were sometimes incomplete or unavailable, which may have compromised the accurate categorization of cheetahs according to health status and disease type. Nevertheless, the apparently unhealthy cheetahs had significantly increased a2-globulin and Hp concentrations, which exceeded the reference ranges for domestic cats. Consequently, the calculated ratio of albumin to globulins was significantly lower in unhealthy cheetahs. Three cheetahs with severe clinical signs (chronic kidney failure, and abscess formation of the anal sac) showed an increase of up to five times the mean Hp concentration (13.8–15.3 g/L), whereas it is known that Hp can increase up to 10 times in diseased domestic cats.29 In addition, cheetahs suffering from chronic kidney disease had significantly elevated a2-globulin and Hp concentrations compared with cheetahs from the research group that were reported to be healthy. These data suggest that a2-globulin, and in particular Hp, are important elevated proteins in this chronic disease, which warrants further attention. The b-globulins comprise the negative APP transferrin, as well as lipoproteins and complement.27 In contrast to the b2 fraction, b1-globulin, which is composed mostly of transferrin, was detected in higher concentrations in healthy cheetahs than reported in cats,35 which might indicate differences between species within the Felidae family. Elevations in the c-globulin concentrations are linked with chronic antigenic stimulation because this fraction mainly consists of immunoglobulins. Irrespective of health status, no significant differences were apparent in b- and c-globulin concentrations in the captive cheetah population. However, healthy older cheetahs had

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elevated c-globulins compared with healthy cheetahs between 2 and 5 yr of age. In senior domestic cats, elevated IgM and IgA concentrations were observed, whereas the APP and complement were unaltered compared with younger cats.4 The higher c-globulin concentrations in senior cats, and possibly cheetahs, are likely a normal phenomenon of aging and not indicative for the dysregulation of the humoral response, as indicated in humans.1 Western blot analysis with cheetah serum revealed that the monoclonal anti-human SAA antibodies used in the LAT targeted the molecular weight of SAA and thus can be used for detection of SAA in serum from captive cheetahs. This confirms the specificity of the assay in the detection of species-specific SAA. In the domestic cat, SAA is described as a major APP and can increase more than 10 times during the acute phase reaction.29 Although SAA is an APP, it can also be increased in chronic disease, such as reactive amyloidosis.19 Although the pathologic pathways involved are not completely understood, a continuing high concentration of SAA is believed to be involved in the pathogenesis of amyloidosis, by damaging deposition of amyloid protein A.14 Reactive amyloidosis is a well-known disease in captive cheetahs and black-footed cats (Felis nigripes) and is linked to renal failure,30,36 thereby highlighting the potential diagnostic value of SAA for exotic felids. The SAA concentration of cheetahs classified as healthy were within the reference range for healthy domestic cats.14 Within Felidae, domestic cats and cheetahs are different species, and different techniques have been used for SAA determination. Hence, comparisons of SAA values between domestic cats and cheetahs might be prone to unvalidated extrapolation. Nevertheless, in accordance with a2-globulin and HP, SAA concentrations of apparently unhealthy cheetahs were significantly higher than those of healthy individuals and exceeded the references ranges of domestic cats, which suggests that SAA is a major APP in the cheetah. Four animals confirmed to be severely ill (chronic kidney failure, severe abscess formation of the anal sac, anorexia, and lateral decubitus) showed an increase of more than 10–50 times the mean concentration (391–1,122 mg/L), which is comparable to SAA concentrations detected in diseased domestic cats.29 Moreover, cheetahs with chronic kidney disease had significantly higher SAA concentrations compared with cheetahs from the research group. This highlights the importance of pursuing further research on kid-

ney failure and reactive amyloidosis in the cheetah. In addition, because amyloidosis in cheetahs is linked to renal failure30 and cheetahs with kidney failure showed a significant increase in both SAA and a2-globulin, it is of interest to increase the knowledge of a2-macroglobulin in cheetahs. This macroglobulin is reported to increase during nephrotic syndrome in cats, which can be caused by amyloidosis, and results in proteinuria, hypoalbuminemia, hypercholesterolemia, and finally renal failure.8,26 CRP has the ability to bind with phosphocholine. Based on this characteristic, Tugiramana et al.37 developed a functional turbidimetrical assay for the determination of CRP in humans. This test was specifically created for developing countries because it is inexpensive and easily applied. However, because this nonimmunologic assay is not dependent on common antigenic sites, it is most likely also useful for other species. Recently, this functional CRP test was screened in horses and demonstrated its species-independent reliability.38 The concentrations detected in this present study of captive cheetahs were not comparable with those previously reported in the healthy domestic cat, which were analyzed by a single radial immunodiffusion assay (172– 204 mg/L)14 and are extremely high compared with values reported in healthy dogs (0.8–22.6 mg/L).28 However, in the domestic cat, CRP is found to be poorly responsive during the acutephase reaction,14 which is in line with the absence of a significant health effect on CRP concentrations in cheetahs. Albumin is the most abundant protein in blood and is the major band observed in serum electrophoresis. As in other mammals, albumin in cats is regarded as a negative APP, because it has been found to fall during the acute-phase response.5 Despite the differences in concentration of positive APP in the cheetah population studied here, no significant difference in albumin concentration between cheetah categories was present.

CONCLUSIONS Results indicate that serum proteins in the cheetah are measurable by routine capillary electrophoresis used in human and veterinary medicine. This study presents a range of serum protein concentrations in a sample of captive cheetahs in which the presence of unknown miscellaneous diseases could not be ruled out. Nevertheless, significant increases in a2-globulins were observed in the apparently unhealthy cheetahs studied here. Furthermore, APP in the

DEPAUW ET AL.—SERUM PROTEIN ANALYSES IN THE CHEETAH

cheetahs can be measured using available immunoassays or non–species-specific techniques, as presented here, which may also be applicable in other exotic felids. In addition, the detected concentrations of SAA and Hp in the cheetah population studied here suggest that SAA and Hp are important APP in diseased cheetahs, particularly in animals with chronic kidney disease. These preliminary data offer a platform for further research into serum proteins and APP during health and disease in the cheetah and possibly other exotic felids. Acknowledgments: This study was funded by the Institute for Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), within the scope of the postgraduate study of the first author (grant number 81219). The authors gratefully acknowledge all zoologic institutions for the donation of cheetah serum: Zoo de Barcelona, Bora˚s Zoo, Breeding Centre for Endangered Arabian Wildlife, Budakeszi Vadaspark, Munster Zoo, Parc et Chateau de Thoiry, Ree Park Ebeltoft Safari, Rostock Zoo, Tisch Family Zoological Gardens. A special thanks to Jacques Kaandorp, Lars Versteege, Paul Vercammen, and Ann Pas for their support during sample collection.

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