ACUTE-PHASE RESPONSES IN HEALTHY AND DISEASED RHESUS MACAQUES (MACACA MULATTA) Author(s): Anne K. H. Krogh, D.V.M., Jo F. H. Lundsgaard, D.V.M., Jaco Bakker, D.V.M., Jan A. M. Langermans, Ph.D., Frank A. W. Verreck, Ph.D., Mads Kjelgaard-Hansen, D.V.M., Ph.D., Stine Jacobsen, D.V.M., Ph.D., and Mads Frost Bertelsen, D.V.M., D.V.Sc., Dipl. A.C.Z.M., Dipl. E.C.Z.M. Source: Journal of Zoo and Wildlife Medicine, 45(2):306-314. 2014. Published By: American Association of Zoo Veterinarians DOI: URL:

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Journal of Zoo and Wildlife Medicine 45(2): 306–314, 2014 Copyright 2014 by American Association of Zoo Veterinarians

ACUTE-PHASE RESPONSES IN HEALTHY AND DISEASED RHESUS MACAQUES (MACACA MULATTA) Anne K. H. Krogh, D.V.M., Jo F. H. Lundsgaard, D.V.M., Jaco Bakker, D.V.M., Jan A. M. Langermans, Ph.D., Frank A. W. Verreck, Ph.D., Mads Kjelgaard-Hansen, D.V.M., Ph.D., Stine Jacobsen, D.V.M., Ph.D., and Mads Frost Bertelsen, D.V.M., D.V.Sc., Dipl. A.C.Z.M., Dipl. E.C.Z.M.

Abstract: Five acute-phase reactants—serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron—were measured using commercially available assays in 110 healthy rhesus macaques (Macaca mulatta), and reference intervals were established for future use in health monitoring of this species. Reference intervals established were as follows: SAA, 29.5–87.7 mg/L; CRP, 0–17.5 mg/L; haptoglobin, 354.3–2,414.7 mg/ L; albumin, 36.1–53.0 g/L; and iron, 13.3–40.2 lmol/L. Furthermore, changes in the acute-phase reactants were studied in two additional groups of animals: eight rhesus macaques suffering from acute traumatic injuries and nine rhesus macaques experimentally infected with Mycobacterium tuberculosis reflecting a chronic active inflammation. In animals with inflammation, SAA and haptoglobin concentrations were moderately increased, while CRP increased more than 200-fold. In addition, marked decreases in albumin and iron concentrations were observed. These results show that SAA, CRP, and haptoglobin are positive acute-phase proteins, whereas albumin and iron are negative acute-phase reactants in rhesus macaques. Key words: Acute-phase proteins, inflammation, Macaca mulatta, reference intervals, rhesus macaques.

INTRODUCTION Throughout evolution, the acute-phase response (APR) has been preserved and is documented to provide valuable markers of systemic inflammation in several species.15,32 Acute-phase proteins (APPs) are synthesized in the liver and released into the blood stream upon stimulation by proinflammatory cytokines as an important part of the APR. APPs are defined as proteins with concentration changes of at least 25% upon a systemic inflammatory response.26,31 The serum concentration can either decrease (negative APP) or increase (positive APP).26 APPs are routinely measured in veterinary medicine with speciesspecific variations due to different acute-phase properties and response patterns.3,13,15,17,26 Commonly measured APPs include C-reactive protein (CRP), serum amyloid A (SAA) and haptogloFrom the Department of Veterinary Clinical and Animal Science, University of Copenhagen, Groennegaardsvej 3 st., 1870 Frederiksberg C, Denmark (Krogh, KjelgaardHansen); Center for Zoo and Wild Animal Health, Copenhagen Zoo, Roskildevej 38, 2000 Frederiksberg, Denmark (Lundsgaard, Bertelsen); Animal Science Department, Biomedical Primate Research Centre, Rijswijk, The Netherlands (Bakker, Langermans); Department of Parasitology, Biomedical Primate Research Center, Rijswijk, The Netherlands (Verreck); and Department of Large Animal Sciences, University of Copenhagen, Hoejbakkegaard Alle´ 5, 2630 Taastrup, Denmark (Jacobsen). Correspondence should be directed to Dr. Krogh ([email protected]).

bin.11 Changes in levels of various factors in the blood can also be used as indicators of inflammatory responses, such as induction of hypoalbuminemia41 or iron deficiency.33 Wild animals in zoologic settings often suppress clinical signs of illness, making them difficult to diagnose properly. Increasing the understanding of inflammatory-induced APRs in veterinary health monitoring could therefore be of great value in zoo species.4,5 Only limited information on APPs in nondomestic animals exist.4,5,25,37 The objective of this study was to establish reference values of SAA, CRP, haptoglobin, albumin, and iron as representatives of APR in healthy rhesus macaques and establish possible correlations with sex and age. Furthermore, the concentrations of these factors in individuals suffering from an acute or chronic active inflammatory condition were measured to investigate their potential as markers for inflammatory responses in rhesus macaques.

MATERIALS AND METHODS Serum samples were obtained from three groups of rhesus macaques. Group 1 consisted of healthy controls and included 110 rhesus macaques kept in free stocks or breeding colonies. Blood was sampled as a part of an annual health check, and only clinically healthy animals with normal blood biochemical and hematologic parameters were included. Group 2 consisted of eight rhesus macaques with acute inflammation caused by various kinds of trauma, primarily bite



wounds from conspecifics. Samples were collected as soon as the keepers noticed the trauma, in most cases within a few hours of the incident. Animals were treated and blood was obtained for health monitoring. Group 3 consisted of nine rhesus macaques suffering from chronic active inflammation after intratracheal inoculation with 1,000 colony forming units (CFU) Mycobacterium tuberculosis as part of another study. Serum samples for this study were collected 24 wk after inoculation. All animals belonged to the Biomedical Primate Research Centre (Rijswijk, The Netherlands). The study was done in accordance with Dutch law and international ethical and scientific standards and guidelines. All blood samples were drawn during ketamine anesthesia (10 mg/kg bodyweight, i.m.). Samples were collected from the vena femoralis into serum tubes (Greiner Bio-one Gmbh, 4550 Kremsmu¨nster, Austria) left for 30 min, and centrifuged at 1,875 g for 10 min. Serum was transferred to polypropylene tubes and frozen within 1 hr of collection and stored upright below 208C for 0 to 17 yr until assayed. All measurements were performed using an automated clinical chemistry analyzer (ADVIA 1800 Chemistry System, Siemens Healthcare Diagnostics, 2750 Ballerup, Denmark) following the manufacturer’s instructions and all measurements were done in duplicate. SAA concentrations were determined using an automated latex agglutination turbidimetric immunoassay based on mouse monoclonal antihuman SAA antibodies (SAA, Eiken Chemical Company, Tokyo 110-8408, Japan) as described for feline, canine, and equine samples.10 The assay was calibrated using heterologous calibration material (human recombinant SAA, Eiken Chemical Company, Tokyo 110-8408, Japan). A commercially available human turbidimetric immunoassay based on anti-human CRP goat polyclonal antibodies was used to determine CRP concentrations (CRP, Randox Laboratories Ltd., London EC2M 7 EA, United Kingdom). Calibration curves were made using a canine CRP calibrator as described for canine samples.27,28 Haptoglobin was determined using a commercially available colorimetric assay (phase, haptoglobin assay, Tridelta Development Ltd., Maynooth, County Kildare, Ireland). Calibration was performed using a calibrator set from the same manufacturer.43 Absorbance measured by spectrophotometry was used to determine albumin and iron concentrations. Bromocresol green (albumin, Siemens


Healthcare Diagnostics, 2750 Ballerup, Denmark) was used to determine albumin concentrations14 and ferrozine (Iron_2, Siemens Healthcare Diagnostics, 2750 Ballerup, Denmark) to determine iron concentrations.1 For all analytes the analyses were performed in duplicate within one analytical run, and observed intra-assay imprecision was estimated by a pooled variance estimate based on the duplicate determinations of healthy individuals (coefficient of variation [CV] ¼ [mean variance]½/overall mean). The results were analyzed using GraphPad Prism version 4.01 for Windows (GraphPad Software, San Diego, California 92037, USA). Reference intervals, including 95% confidence interval (CI) plus 90% CI of limits, were calculated by the use of Reference Value Advisor V2.1,18 and assessed according to human and veterinary guidelines.16,21 The data were evaluated by calculating medians, SDs, minima, and maxima, and were visually assessed by depicting histograms. Outliers were detected by Tukey’s method, which defines suspected outliers as 1.5 times the likely range of variation above or below the third or first quartiles respectively, and outliers as three times the likely range of variation above or under the quartiles. Distribution of data was evaluated using the D’Agostino and Pearson omnibus normality test. Reference intervals were computed as nonparametric. To evaluate if there was a difference between the median of groups of diseased animals and control animals, Kruskal-Wallis tests were performed and further characterization of the difference was done by Dunn’s multiple comparison test. Any difference in concentration of acutephase reactants between males and females was evaluated by a Mann-Whitney test. If a difference was detected, the need for possible partitioning was assessed according to CLSI C28-A3.21 The significance level was set to P , 0.05 in all tests. To evaluate if the age of the animal was related to the concentrations of APPs, a linear regression analysis was performed, where a slope different from zero would indicate correlation. Finally, to evaluate a possible significant influence of storage time, a linear regression analysis was performed on the measured concentrations in healthy individuals according to time of storage, where influence is assessed as minimal if no correlation could be observed.

RESULTS Data as well as the reference intervals are summarized in Table 1. The width of the 90%


Table 1.


Reference intervals and descriptive data for five acute-phase reactants in rhesus macaques.

Acute-phase reactanta


SAA (mg/L) CRP (mg/L) Haptoglobin (mg/L) Albumin (g/L) Iron (lmol/L)

97 110 110 105 105

a b

Median 6 SD

47.8 0.2 987.8 44.8 26.3

6 6 6 6 6

12 4.4 535.3 4.1 6.2



Reference interval

25.7 0 286.7 32 12.1

90.1 33.4 2,452 60.1 43.4

29.5–87.7 0–17.5 354.3–2,414.7 36.1–53.0 13.3–40.2

SAA, serum amyloid A; CRP, C-reactive protein. Varied number of animals included due to insufficient sample material.

CI for the upper or lower limit was wider than 20% of the range of the reference interval for all APPs except for haptoglobin. Outliers detected according to Tukey showed three and one suspected outliers for SAA and iron, respectively. Albumin showed three suspected outliers and one outlier. The data were not excluded. Levels of SAA, albumin, and iron were not different between males and females. However, males and females showed statistically significant differences in their serum CRP levels (females: median ¼ 0.15 mg/L, males: median ¼ 0.4 mg/L; P ¼ 0.025) and haptoglobin levels (females: median ¼ 939.1 mg/L, males: median ¼ 1,415 mg/L; P ¼ 0.01) (Fig. 1). Intra-assay imprecision (CV) was assessed to be 10%, 18%, 2%, 2%, and 2% for SAA, CRP, albumin, haptoglobin, and iron, respectively. No correlation between age and CRP and iron concentrations was determined. There was, however, a correlation between age and SAA, albumin, and haptoglobin concentrations, with slopes of 0.52 mg/L/yr (95% CI, 0.09 to 0.95), 0.16 g/ L/yr (95% CI, 0.30 to 0.01), and 26.45 mg/L/yr (95% CI, 8.44 to 44.46), respectively (Fig. 2). No

correlation between measured concentrations and time of storage was observed for CRP, albumin, haptoglobin, and iron. For SAA a mild linear correlation (R2 ¼ 0.12, P , 0.05) was observed with a positive regression slope of 0.9 mg/L/yr. Animals with acute inflammation had higher serum concentration of CRP (P , 0.01) and lower serum concentrations of albumin (P , 0.001) and iron (P , 0.01) compared to healthy animals. The serum concentrations of SAA and haptoglobin showed no statistically significant difference. In animals suffering from chronic active inflammation, a statistically significant higher concentration (P , 0.001) of serum concentrations of SAA, CRP, and haptoglobin were determined as well as a lower concentration of albumin and iron (P , 0.001) (Fig. 3).

DISCUSSION In the present study, reference intervals for SAA, CRP, haptoglobin, albumin, and iron were established in rhesus macaques. For all APPs, the 90% CI of the upper or lower limit were larger than recommended in IFCC-CLSI C28-A3.21

Figure 1. C-reactive protein (CRP) and haptoglobin serum concentrations in healthy female and male rhesus macaques. The horizontal lines represent median concentrations. The median concentration between female and male varied significantly (P , 0.05) for (a) CRP and (b) haptoglobin.



Figure 2. Correlation between age and serum concentrations of serum amyloid A, albumin, and haptoglobin in healthy rhesus macaques displaying slopes deviating significantly from zero. The lines represent the correlation between age and the concentration of acute-phase protein.

The CRP determined concentrations in rhesus macaques correspond well with two other studies performed in long-tailed macaques (Macaca fascicularis).22,23 The median values for haptoglobin are also similar to those found in studies in other species (including humans), where normal values reached no more than 3,000 mg/L.7,25,34,38,42 Previous studies of albumin concentrations in rhesus macaques have shown means of 39.5 6 4.8 g/L SD; 33 6 3 g/L SD to 47.4 6 4 g/L SD; and 53.87 6 2.67 g/L SD,6,8,40 and the findings in this study correlate well with these values. Finally, another study of rhesus macaques showed that the mean value of iron was 27.98 6 5.31 lmol/L SD,8 which also corresponds to the results obtained in this study, and with studies in long-tailed macaques.19,35 Collectively, this shows that the assays

used in this study are valid to determine APR values in macaques. The results showed no significant sex-associated variations in SAA, albumin, and iron concentrations. CRP and haptoglobin showed a significantly higher concentration in males compared to females, but following the recommendations of American Society for Veterinary Clinical Pathology (ASVCP) reference interval guidelines16 no further reference interval partitioning was necessary due to the difference between medians of males and females being less than 25% compared to range of the reference interval.16 Overall, these data suggest that gender has no marked effect, which is in accordance with studies in other species.9,19,22,24,30,35 The study showed a correlation between age and SAA, albumin, and haptoglobin concentra-



Figure 3. (a) Serum amyloid A (SAA), (b) C-reactive protein (CRP), (c) albumin, (d) haptoglobin, and (e) iron serum concentrations in three different groups of rhesus macaques. The horizontal black lines represent the median values in the respective groups and the dashed lines represent the upper or lower limit of the reference interval for the specific acute-phase reactants established in this study. The significance level of difference between the median values of the groups compared to control is noted above the relevant groups. (þ) n ¼ 5, due to insufficient sample material.

tions with slopes (95% CI) of 0.52 mg/L/yr (0.09 to 0.95), 0.16 g/L/yr (0.3 to 0.01), and 26.45 mg/L/yr (8.44 to 44.46), respectively. This indicates that SAA and haptoglobin concentrations increase whereas the albumin concentration decreases with age in healthy individuals. The

results for albumin correlate well with earlier studies in rhesus macaques and humans.6,38,40 It has been suggested that changes in APR can be due to altered cytokine regulation.2,38 The changes across age are, however, considered to be minute compared to APR-induced changes, and only


when comparing extreme age groups will the agedependent difference approach the criteria of representing 25% of the range of the reference interval, rendering reference interval partitioning with age clinically irrelevant. When comparing APR concentrations in healthy animals with those in animals with acute and chronic inflammation, all investigated markers showed acute-phase properties. In animals with acute inflammation, CRP was significantly higher (P , 0.01) and albumin ( P , 0.001) and iron (P , 0.01) significantly lower. In animals with chronic active inflammation the concentrations of SAA, CRP, and haptoglobin (P , 0.001) were higher while albumin and iron concentrations (P , 0.001) were lower than in healthy animals. SAA is a major APP in several species of animals,26 and it has been shown to increase up to 1,523% in an experimental study with longtailed macaques administrated with a dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).12 Present data showed a 368% increase in chronic active inflammation and only a slight increase in the group of animals suffering from acute inflammation, which does not correspond well with its status as a major APP. This could be due to a low cross-reactivity of the monoclonal antibodies in the assay used in this study, resulting in a low analytical sensitivity. We were, however, able to detect marked elevated concentrations (up to 1,539 mg/L, data not shown) in a rhesus macaque 4 wk after inoculation with M. tuberculosis. Alternatively—and more likely—the SAA concentration had not yet peaked, since blood sampling was performed only a few hours after the acute insults. Concentrations of major APPs typically increase within 6–12 hr of an acute inflammatory insult, and peak concentrations occur after 24–36 hr.26,36 These uncertainties warrant continued study. CRP is a major APP in humans, macaques, dogs, rats, pigs, and rabbits.26,36,44 In dogs and humans, concentrations vary according to severity of trauma.20,46 In this study, CRP showed a dramatic increase for some individuals and an overall median 295-fold increase in the group with acute inflammation. This corresponds very well with the expected profile of a major APP, with low values in normal healthy individuals and a severely increased concentration upon inflammation. Haptoglobin is described in several species as a moderate APP with a less than 10-fold increase in response to inflammation.15 In this study the median concentration of haptoglobin showed a


twofold increase in chronic active inflammation. In a previous study with long-tailed macaques administrated with a dopaminergic neurotoxin (MPTP), mean haptoglobin concentrations increased threefold.12 This difference could be due to variation in tissue injury. The results suggest that haptoglobin acts as a moderate APR in rhesus macaques. However, nearly all values of the diseased animals are within the reference interval computed in this study, indicating that the diagnostic potential of haptoglobin is limited. Albumin is an indicator for an APR in several domesticated animals.36 In a toxicology study performed in long-tailed macaques, albumin serum concentration decreased between 12% and 49% dependent on dosage of an immunotoxin.29 The decline in concentration in this study was 14.6% and 21% in the groups of acute inflammation and chronic active inflammation, respectively. The decreased concentration in the acute inflammatory group could also be influenced by acute blood loss and increased permeability of vessels induced by inflammation. Iron has several roles and functions within the body. In the APR, plasma concentrations of iron decline rapidly, reflecting sequestration of iron in the cytoplasm of macrophages and enterocytes due to increased hepcidin production.33 The degree of trauma corresponds with the concentration of iron.45,47,48 A 46% reduction of iron documented in long-tailed macaques with IL-6 induced inflammation39 corresponds to the 42% and 67.6% reductions found in this study. Overall, iron acts as a negative acute-phase reactant in rhesus macaques. CRP and haptoglobin showed differences in median serum concentrations between sexes, but further partitioning was not required due to a ,25% difference between medians of males and females compared to the ASVCP reference interval guidelines.16 SAA, haptoglobin, and albumin concentrations showed correlation to age of the animals. The concentrations of SAA, CRP, and haptoglobin increased (positive APPs) and concentrations of albumin and iron decreased (negative acute-phase reactants) in response to systemic inflammatory stimuli. SAA has been shown to be a major APP in several species26 including long-tailed macaques.12 Here, SAA concentrations showed a reference interval of 29.5 to 87.7 mg/L and a median of 47.75 mg/L. There were three suspected outliers concentrated in the upper limit of the range when calculated according to Tukey, suggesting that these individuals may not have been healthy and



could have suffered from a subclinical systemic inflammatory response. They were, however, retained in the study as recommended by IFCCCLSI21 because they fulfilled original inclusion criteria. The range of the 90% CI of the upper and lower limits was larger than recommended by IFCC-CLSI,21 indicating that uncertainty may be critical. Importantly, however, the limits of the 90% CI are included in the final reference interval to account for this. Later studies enrolling lager sample sizes may limit this uncertainty. There are several limitations to the study. The extended time of storage could influence the measured concentrations of the analytes included. Only for SAA was such a correlation observed, with an average increase of 0.9 mg/L/yr, which indicates degradation has not occurred; further, the increase is of limited significance compared to the major increases observed in positive cases with inflammatory activity, thus it was not expected to compromise diagnostic sensitivity by biasing the assessed reference limit. Finally, the healthy individuals included represents the normal variation of several clinical variables, including body condition score (BCS), which was, however, not registered and tested for influence on results. However, it is the authors’ opinion that BCS within normal range is an integral and expected part of the variation represented by reference limits. In summary, this study established reference intervals of SAA, CRP, haptoglobin, albumin, and iron in rhesus macaques and further described CRP as a major APP, SAA and haptoglobin as moderate APPs and albumin and iron as negative acute-phase reactants based on acute traumatic injuries and chronic infection with M. tuberculosis. Monitoring of systemic inflammatory activity in this species can benefit from these reference intervals. The study indicates that it is possible to use commercially available automated assays to measure acute-phase reactants in rhesus macaques. Acknowledgments: A special thanks to Kerstin Ma¨tz-Rensing, Deutsches Primatenzentrum GmbH, Go¨ttingen, Germany, and the staff at the Central Laboratory, Department of Veterinary Clinical and Animal Science, University of Copenhagen, Denmark. Parts of the study were funded through a donation from Alfred Benzon’s Foundation to Copenhagen Zoo, Denmark, and in part by the European Union (EUPRIM-NET2, grant agreement 262443).

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Acute-phase responses in healthy and diseased rhesus macaques (Macaca mulatta).

Five acute-phase reactants-serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron-were measured using commercially available ...
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