Comp Clin Pathol DOI 10.1007/s00580-012-1492-8
ORIGINAL ARTICLE
Acute phase protein profile in agoutis (Dasyprocta azarae; Lichtenstein, 1823) in captivity, determined by sodium dodecyl sulfate polyacrilamyde gel electrophoresis Elizabeth Moreira dos Santos Schmidt & Rogério Ribas Lange & Érika Fruhvald & Ivan Felismino Charas do Santos & José Jurandir Fagliari
Received: 30 November 2011 / Accepted: 11 April 2012 # Springer-Verlag London Limited 2012
Abstract The acute phase response refers to a nonspecific and complex systemic reaction of the organism that occurs shortly after any tissue injury. The acute phase response is considered a part of the innate host defense system, which is responsible for the survival of the host during the critical early stages of attack, and in evolutionary terms, it precedes the acquired immune response. The purpose of this study was to determine serum protein concentrations, including the acute phase protein profile in agoutis (Dasyprocta azarae) in captivity, by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blood samples from 11 adult healthy animals (nine females and two males) were obtained. The serum proteinogram had 21 proteins with molecular weights ranging from 15 to 240 kD. The acute E. M. dos Santos Schmidt (*) : É. Fruhvald Department of Veterinary Clinics, São Paulo State University “Júlio de Mesquita Filho” (UNESP), Botucatu, São Paulo, Brazil e-mail:
[email protected] E. M. dos Santos Schmidt e-mail:
[email protected] R. R. Lange Department of Veterinary Medicine, Federal University of Paraná State, Curitiba, Paraná, Brazil I. F. C. do Santos Department of Veterinary Surgery and Anesthesiology, São Paulo State University “Júlio de Mesquita Filho” (UNESP), Botucatu, São Paulo, Brazil J. J. Fagliari Department of Veterinary Clinics and Surgery, São Paulo State University “Júlio de Mesquita Filho” (UNESP), Jaboticabal, São Paulo, Brazil
phase proteins identified were: ceruloplasmin, transferrin, albumin, haptoglobin, α-1-acid glycoprotein, and hemoglobin. IgA, IgG heavy and light chains, and nonnominal identified proteins of 240, 210, 140, 98, 78, 48, 35, 31, 23, and 15 kD were also identified. The determination of the acute phase protein concentrations is a useful method for the early detection of subclinical disease or changes in the healthy animal, with predictive information on the development of disease in the future. It is possible to standardize the reference values of the serum protein profile of agoutis, which can be used for diagnosis and prognosis, treatment and clinical follow-up of nutritional disorders, and immunemediated inflammatory diseases that may affect these animals. Keywords Acute phase proteins . Agoutis . Dasyprocta azarae . Electrophoresis . SDS-PAGE
Introduction Acute phase response is the term used to describe a prominent systemic reaction of the organism with events like vasodilation; platelet aggregation; neutrophil chemotaxis; lysosomal enzymes release; histamine release and oxidative radicals release; fever; increased levels of cortisol; decreased thyroxine concentrations; leukocytosis; thrombocytosis; muscle catabolism; gluconeogenesis; decrease of iron, calcium, and zinc; and changes in hepatic synthesis of acute phase protein (APP) with the inflammatory process (van Leeuwen and van Rijswijk 1994; Cerón et al. 2005; Gruys et al. 2005). The main action of the acute phase response is to facilitate the inflammation and repair process and to protect the body from
Comp Clin Pathol
the destructive potential of inflammatory products and to restore homeostasis (Eckersall 2008). This response occurs in chronic inflammation and in acute processes and is characterized by a fast and nonspecific response, which occurs prior to the stimulation of specific immune response and can be observed before the clinical signs (van Leeuwen and van Rijswijk 1994). The APP production differs between species and it is produced mainly by hepatocytes and controlled by proinflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor alpha, released by macrophages from the site of pathogenic or inflammatory tissue injury (Cerón et al. 2005; Gruys et al. 2005; Eckersall 2008). In several combinations of species and assay, significantly different levels of proteins were detected between healthy individuals and individuals with an acute phase response, suggesting that specific proteins acted as an APP and the applied assay possessed detective abilities for those proteins (Bertelsen et al. 2009). The rodents belong to a group of 2,277 species that are recognized, representing 42 % of all biodiversity of mammals in the world (Tirira 1999; Wilson and Roeder 2011). The agouti (Dasyprocta azarae), which belongs to the Rodentia order, is a small rodent that is distributed throughout the Brazilian territory. It belongs to the Stricognatha suborder, as well as the pacas, capybara, chinchillas, porcupines, among other species. Characteristics of Dasyprocta sp. includes a robust head, large eyes and small ears, long and curved back, an obsolete tail without hair, and can weigh 3 to 6 kg and reach 18 years old (Oliveira and Bonvicino 2006; Lange and Schmidt 2007). These animals have morning and crepuscular and ground habits. The diet of these animals comprises fruits, seeds, and roots (Voss and Emmons 1996; Oliveira and Bonvicino 2006). Despite the D. azarae be on the list of the animals with low risk of extinction, the species Dasyprocta leporina (Linnaeus 1766) is on the list of animals with medium extinction in São Paulo State, Brazil (Bressan 2009). Because of many similarities between this two species and due to the fact that both belong to the same genus, reference values for one may be useful for the clinical use of the other. Reference values of acute phase proteins profile for wildlife animals are rare, especially in animals from South America. Regarding to the wildlife, the literature reports the APP profile by means of sodium dodecyl sulfate polyacrilamyde gel electrophoresis (SDS-PAGE) to giant anteaters (Myrmecophaga tridactyla), gray brocket deer (Mazama gouazoubira) and paca (Agouti paca) (Satake and Fagliari 2006; Santana et al. 2008). By determining these values, it is possible to evaluate the animals regarding their hydration status, presence of inflammation, infection, immunemediated disease, and protein synthesis (Gruys et al. 2005). To check alterations of APP concentrations in sick animals, it is necessary to determine references parameters in healthy animals. This type of data is not
available for agoutis in the literature. In that way, the aim of this study is to determine the serum protein concentration, including the APP profile, by means of SDS-PAGE for the species D. azarae, in captivity.
Material and methods Eleven healthy adults agoutis (D. azarae) were used, two males and nine females. The health status was evaluated by clinical examination and hemograms. They belonged to the scientific breeding of wild animals of the Natural History Museum Capão of Imbuia, Paraná, Brazil (25 ° 25′ S and 49 ° 16′ W), with the same management regarding to the food and other habits. The animals were fasted for 8 h and physically restrained according to the routine for blood collection. The blood was collected from the lateral saphenous vein, according to Pachaly et al. (2001), and stored in tubes without anticoagulant to obtain serum samples. The concentrations of total serum proteins were determined by the biuret method, using a commercial kit (Diagnostica Labtest®, Lagoa Santa, Minas Gerais, Brazil) with a semiautomatic spectrophotometer (Labquest, Labtest®, Lagoa Santa, Minas Gerais, Brazil). Protein electrophoresis fractionation was determined by means of polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE), as recommended by Weber and Osborn (1969) and the gel separation and polymerization as recommended by Fagliari et al. (1998). The videodensitometry (CS 9301® Shimadzu Scientific Instrument, Kyoto, Japan) was used to determine the molecular weights and protein concentrations fractions. Reference markers (Sigma-Aldrich®, St. Louis, USA) were used with molecular weights of 20, 24, 29, 45, 55, 66, 97, 116, and 200 kD to identify the proteins. Based on densitometric evaluation of protein bands, it was possible to make reference bends from the reference marker and by comparison with electrophoretic mobility of purified IgA, ceruloplasmin, transferrin, albumin, haptoglobin, α-1-acid glycoprotein, IgG, and 23 kD. The GraphPad Prism 5® statistical program was used, and the minimal and maximum values from each protein fraction were analyzed using mean and standard deviation.
Results For all animals, the serum protein electrophoretograms were similar. The molecular weight for each protein, the average, the standard deviation, and the minimal and maximum values of total serum protein concentration, albumin, and the other protein fractions are shown on Table 1. The total serum protein and albumin results were presented in grams per deciliter and the other protein fraction in milligrams per deciliter. The total serum protein mean was 6.9 g/dL.
Comp Clin Pathol Table 1 Total serum protein concentration (g/dL), albumin (g/dL), and protein fractions (mg/dL) by means of SDSPAGE, from agoutis (D. azarae), in captivity, in Brazil
Proteins
Molecular weight (kD)
Maximum values
5.76
7.64
240
12.5±13.5
0a
44
NI IgA
210 165
24±12.7 360±63
7.3 241
NI Ceruloplasmin NI Transferrin
140 117 98 90
17.6±18.5 10±8 81±33.9 490±37
0a 0a 28.9 427
42.6 462 61.6 28
NI Albumin IgG heavy chain
78 65 54
22.1±21 3.8±0.3 1,030.4±142.7
0a 3.3 780.3
NI
48
37.2±15
12.7
61.3
Agoutis (n011), in captivity, from the Natural History Museum Capão of Imbuia, Curitiba, Paraná, Brazil, were physically restrained
Haptoglobin α-1-acid glycoprotein
41 37
150±68 100±31
47 34
NI NI
35 31
48.3±18.8 50.2±28.9
0a 18.4
230 147 67.3 97
NI nonnominal identified protein
IgG light chain NI Hemoglobin
29 23 17
1,070±202 380±110 73.3±40.2
693 238 18.5
1,389 586 144.6
NI
15
132.1±95.3
24.7
279
Two animals did not show the NI (240 kD), ceruloplasmin, and NI (78 kD) and NI (35 kD) fractions
6.9±0.5
Minimal values
NI
a
Total serum protein
Mean ± SD
The SDS-PAGE technique allowed the fractionation of 21 serum proteins ranging from a molecular weight of 15 to Fig. 1 Electrophoretogram was obtained from the densitometric evaluation of an individual protein band (D. azarae). Peaks 1 240 kD NI protein, 2 210 kD NI protein, 3 IgA, 4 140 kD NI protein, 5 ceruloplasmin, 6 98 kD NI protein, 7 transferrin, 8 78 kD NI protein, 9 albumin, 10 IgG heavy chain, 11 48 kD NI protein, 12 haptoglobin, 13 α-1-acid glycoprotein, 14 35 kD NI protein, 16 31 kD NI protein, 18 IgG light chain, 19 23 kD NI protein, 20 hemoglobin, 21 15 kD NI protein. NI nonnominal identified protein
146.3 547 65.1 4.1 1,195.9
240 kD, which were identified in all agoutis. It was possible to identify nine protein fractions: IgA, ceruloplasmin,
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transferrin, albumin, haptoglobin, α-1-acid glycoprotein, IgG heavy and light chains, and nonnominal identified proteins of 240, 210, 140, 98, 78, 48, 35, 31, 23, and 15 kD were identified. Two animals did not show the fractions of 240 kD, ceruloplasmin, and 78 and 35 kD (Fig. 1). Although the electrophoresis showed 21 proteins, there are purified reference markers (standards) available that allowed the identification of only nine of them.
Discussion The electrophoretic fractionation is a simple and inexpensive technique. It is one of the most reliable methods of blood proteins identification. It can detect 20 to 30 different fractions, including immunoglobulins and APP, in extremely low protein concentration (Gordon 1995). Studies have shown that the increase of APP concentration can occur by physical or physiological stress during animal transport and this increase and duration are directly proportional to the quality and length of stress exposure and may be an important parameter, which in the future can be used to measure animal welfare (Piñeiro et al. 2007). Total serum protein mean was 6.9 g/dL. Lange and Schmidt (2007) reported similar values for total serum protein in this species. In the agouti serum proteinograms obtained in polyacrylamide gel containing sodium dodecyl sulfate, nine proteins were identified nominally, and eight were only identified by their molecular weights, similar to the eight protein fractions identified in pacas, but lower than the 34 fractions observed in gray brocket deer and 47 fractions found in giant anteaters (Voss and Emmons 1996; Santana et al. 2008). This study showed the presence of the α-1-acid glycoprotein, not observed in pacas. On the other hand, the α-1antitrypsin was identified in paca, but not in agoutis. Differently from that observed in agoutis, which showed nonnominal proteins with 98, 78, 23, and 15 kD, the pacas showed different nonnominal proteins. However, the pacas also showed the 240, 210, 35, and 31 kD fractions (Santana et al. 2008). Even so that paca belongs to the Rodentia order and is a typical animal of tropical regions like agoutis, there are differences in the serum protein profile (Lange and Schmidt 2007). Thus, due to variation in protein fractions observed in wild animals, further investigations are needed to determine the protein electrophoresis profile of each species as well studies to determine the structure and function of those proteins not identified nominally. The determination of the APP concentrations is a useful method for the early detection of subclinical disease or changes in the healthy animal, with predictive information on the development of disease in the future. Changes in serum concentrations of these proteins indicate the necessity for a detailed assessment of
the animal, besides being a way to monitor management and treatment of the patient.
Conclusion In this study, all animals belonged to the same group and underwent to the same management, including nutrition. On this way, it is possible to standardize the reference values of the serum protein profile of this species, which can be used for diagnosis and prognosis, treatment and clinical follow-up of nutritional disorders, and immune-mediated inflammatory diseases that may affect these animals.
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