Plasma Protein Electrophoresis as a Diagnostic Tool for Endangered Asian Reptiles
World Small Animal Veterinary Association World Congress Proceedings, 2015
Z. Knotek1, A. Musilova1, K. Pinterova2, Z. Knotkova1
1Avian and Exotic Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic; 2synlab czech s.r.o., Brno, Czech Republic

Professor Zdenek Knotek (Sid) is the head of the Avian and Exotic Animal Clinic on Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic. As a visiting professor Sid had part of his teaching activities dealing with reptiles in other universities (Budapest, Denpasar, Copenhagen, Kosice, Ljubljana, Purdue [IN, USA], Utrecht, Vienna, Warsaw). He is a founding member and diplomate of the European College of Zoological Medicine (herpetology), member of ARAV, a past-president of the European Association of Zoo and Wildlife Veterinarians (EAZWV), and a president of the Czech Association of Zoo and Wildlife Veterinarians (CAZWV). As a founder of reptile medicine specialty at Clinic for Reptile, Avian and Fish Medicine Professor Knotek headed the Unit for Reptiles and Pet Birds at University of Veterinary Medicine Vienna (2010–2013). His current focus involves medicine and surgery in reptiles, small mammals and birds. Sid supervises international training courses for exotic medicine: Summer School for Exotic Medicine and Surgery (since 2004) and ESAVS courses - Exotic Pets Medicine and Surgery I, II, III (since 2005).


Serum or plasma protein electrophoresis is used as a routine test for health assessment in veterinary medicine, including exotic patients. It is an important laboratory tool for diagnosis of infectious diseases, various forms of liver disease and chronic renal failure. As compared with mammals or birds, electrophoresis in reptiles is used rarely in clinical practice. This can be partly caused by the limited availability of reliable data on the physiological composition of individual fractions in reptiles. The electrophoretic values obtained from individual laboratories can vary and the reference intervals are in a way dependent on a diagnostic system used by a given laboratory. The main protein fractions (e.g., albumin or prealbumin, alpha, beta and gamma globulins) have been observed in reptiles, but reference intervals have been published only for a limited number of reptile species.1-7

The aim of this study was to analyse the plasma protein electrophoresis profile in a group of healthy reptiles and some patients. Seven reptiles were included to this study: three healthy Horsfield´s tortoises (Testudo horsfieldii), one tortoise of the same species suffering from chronic anorexia, one terrapin Cistoclemmys (Cuora) flavomarginata suffering from anorexia and hyperuricaemia, one Burmese python female (Python mollurus bivittatus) with stomatitis-tracheitis complex and one Burmese python male (Python mollurus bivittatus) with a chronic adenovirus and paramyxovirus infection.

During the clinical control and treatment all animals of this study were kept at the Avian and Exotic Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic. The animals were housed and handled in agreement with the Branch Commission for Animal Welfare of the Ministry of Agriculture of the Czech Republic (accreditation No. 46613/2003–1020). Animals were kept in terrariums at temperatures ranging from 24 to 35°C and air humidity between 60 and 80%. The animals had a specific light regime (12 h light/12 h dark, 60 W bulb + UV lamp Repti-Glo 5.0, Hagen). The diet of healthy tortoises consisted mainly of dandelion flower leaves with calcium supplement, the tortoise patients were force fed in 2–3 day intervals with a special supportive instant diet (Lafeber´s Emeraid LLC, USA). The terrapin was force fed with the special instant diet (Convalescence Support Canine/Feline, Royal Canin, EU) and received allopurinol (20 mg/kg orally, q 24 h, Milurit 100, Egis Pharmaceuticals Plc, Hungary). Pythons received supportive therapy (antibiotics, fluid therapy with amino acids, minerals, vitamins, glucose). The body condition and health status of animals was checked 1–2 times a day by clinical examinations and blood profiles (haematology and plasma chemistry) were analysed. Tortoises and terrapin were manually restrained and blood samples were collected from the subvertebral plexus. Snakes were manually restrained in dorsal recumbency and blood samples were collected from the ventral coccygeal vein (vena coccygea ventralis). Blood in 2 ml heparinized syringes (50 IU/ml, Heparin Léčiva inj., Prague, Czech Republic) were centrifuged (6440 g for 10 minutes) and plasma samples were kept in a freezer at -20°C until analysis.

Analysis was performed within 1–3 months, with no thawing in between. Hemolytic or lipemic plasma samples were excluded from the analysis. Total protein was determined by the biuret method using Modular Analytics SWA (Roche). Agarose gel electrophoresis was performed on 10 µl plasma samples using an automated Hydrasis system and Hydragel Protein(e) 15/30 agarose gels (Sebia) at 20 W and 20°C for 7 minutes. Gels were stained with Amido Black (Sebia) according to manufacturer's instruction. Densitometer Preference (Sebia) was used for scanning the electrophoretograms. For protein fractionation, the midpoint of the electrophoretogram was first identified, which lies between the α2 and β1 peaks, in order to orientate the peak allocation.8 The product of percentage volume of each fraction and total protein concentration was calculated to determine individual fraction concentration in grams per litre. In tortoises and one terrapin of the present study four main protein fractions have been observed: albumin, alpha globulins, beta globulins and gamma globulins (Table 1). In comparison with healthy tortoises the patient with chronic anorexia had a lower concentration of beta globulins (3.21 vs. 7.52–10.65 g/l) and a higher concentration of gamma globulins (6.15 vs. 2.21–3.87 g/l). The concentrations of total proteins, albumin, alpha globulins and albumin to globulin ration (A/G) in the patient were within the ranges for healthy tortoises (24.9 vs. 23.5–41.6 g/l, 11.93 vs. 10.97–23.30 g/l, 3.61 vs. 2.77–3.79 g/l, 0.92 vs. 0.88–1.27, respectively).

Higher concentrations of total protein, albumin and alpha globulins for Hermann´s tortoises (Testudo hermanni) were published by Fiorucci et al. (2013), the A/G ration was similar with the value observed in healthy Horsfield´s tortoises in the present study.9 The methodologies used for determination of protein fractions in these two studies were different. Fiorucci et al. (2013) analysed the serum with cellulose acetate electrophoresis (CAE). It is always necessary to specify which commercial electrophoretic system is used in the study. For instance, electrophoresis in connection with total protein determination by the biuret method is recommended for determination of albumin in reptile plasma samples.10 The risk of overestimation of the plasma albumin concentrations with the bromocresol green dye-binding method in birds and turtles has been reported.11,12 The method of agarose gel electrophoresis (AGE) for plasma sample analysis used in the present study was used by Andreani et al. (2014) for Hermann´s tortoises.13 They published results with higher plasma concentrations of total protein (40.0 ± 2.0 g/l) and alpha globulins (10.5 ± 0.7 g/l), but very similar concentrations of beta and gamma globulins. Although the methodology of determination of protein fractions could be identical in some studies differences in breeding conditions cannot be excluded. The results that we received for a terrapin patient are similar with the ranges that have been published recently for the Eastern box turtle.14

Four main plasma protein fractions have been observed in two Burmese python patients: albumin, alpha globulins, beta globulins and gamma globulins. In comparison with the results of Silva et al. (2011) snakes of the present study had higher plasma concentrations of albumins and alpha globulins and lower plasma concentration of gamma globulins.7 Variability of the results is to a certain extent conditioned by a significant effect of external factors that affect reptilian metabolism. In a previous study authors observed significant changes of biochemical parameters during the year.7 For instance, there was a significant difference between alpha globulin concentrations established in boa constrictors in the summer and in the winter.7 Some differences can also be caused by different species that have been studied (Pythoninae vs. Boinae). The determination of electrophoretic values for plasma proteins in commonly kept reptiles is becoming a very relevant issue. Further studies are needed to establish reference intervals for other reptile species and to determine diagnostic and prognostic value of plasma protein electrophoresis in diseased animals.

Table 1. Plasma protein fractions in tortoises, terrapin and snakes

Reptiles

Total protein (g/l)

Albumin (g/l)

α globulins (g/l)

β globulins (g/l)

γ globulins (g/l)

A/G

Testudo horsfieldii

31,5

16,32

2,77

9,67

2,74

1,07

Testudo horsfieldii

23,5

10,97

2,80

7,52

2,21

0,88

Testudo horsfieldii

41,6

23,30

3,79

10,65

3,87

1,27

Testudo horsfieldii*

24,9

11,93

3,61

3,21

6,15

0,92

Cistoclemmys flavomarginata

35,7

8,14

7,50

13,35

6,71

0,30

P. mollurus bivittatus

66,9

39,47

15,86

7,69

3,88

1,44

P. mollurus bivittatusa

74,9

43,37

14,61

14,53

2,40

1,38

P. mollurus bivittatusb

54,3

30,84

10,26

11,13

2,06

1,31

A/G albumin/globulin ration
a-b plasma samples of the same snake (time interval 15 days)
* patient

Acknowledgments

This project received partial support from the University of Veterinary and Pharmaceutical Sciences Brno (Specificky vyzkum 2014/2015). Authors would like to thank to Mrs. Petra Majkova for statistical analysis and to Dr. Silvia Barazorda Romero, Dr. Ing. Eva Cermakova and Mrs. Dagmar Karesova for their skillful assistance.

References

1.  Girling SJ. The use of protein electrophoresis in reptiles as an aid to diagnosis. In: Proceedings from the 1st International Conference on Reptile and Amphibian Medicine; March 4–7, 2010: 217–219; Munich, Germany.

2.  Deem SL, Dierenfeld ES, Sounguet GP, et al. Blood values in free-ranging nesting Leatherback sea turtles (Dermochelys coriacea) on the coast of the Republic of Gabon. J Zoo Wildl Med. 2006;37:464–471.

3.  Giménez M, Saco Y, Pato R, Busquets A, Martorell JM, Bassols A. Plasma protein electrophoresis of Trachemys scripta and Iguana iguana. Vet Clin Pathol. 2010;39:227–235.

4.  Cooper-Bailey K, Smith SA, Zimmerman K, Lane R, Raskin RE, DeNardo D. Hematology, leukocyte cytochemical analysis, plasma biochemistry, and plasma electrophoresis of wild-caught and captive-bred Gila monsters (Heloderma suspectum). Vet Clin Pathol. 2011;40:316–323.

5.  Proverbio D, Bagnagatti de Giorgi G, Della Pepa A, et al. Preliminary evaluation of total protein concentration and electrophoretic protein fractions in fresh and frozen serum from wild Horned Vipers (Vipera ammodytes ammodytes). Vet Clin Pathol. 2012;41:582–586.

6.  Müller K, Brunnberg L. Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye-binding method. Vet Clin Pathol. 2010;39:79–82.

7.  Silva LF, Riani-Costa CC, Ramos PR, Takahira RK. Seasonal influence on biochemical profile and serum protein electrophoresis for Boa constrictor amarili in captivity. Braz J Biol. 2011;71:517–520.

8.  Ceron JJ, Caldin M, Martinez-Subiela S. Answers to some common questions on serum protein electrophoresis. Vet Rec. 2011;168:453–454.

9.  Fiorucci L, Ceccarelli M, Macrelli R, Crosta L. Protein electrophoresis in Hermann's tortoises (Testudo hermanni): a first step in reference value determination. J Herpetol Med Surg. 2013;23:15–19.

10. Campbell TW. Clinical pathology of reptiles. In: Mader DR, ed. Reptile Medicine and Surgery. 2nd ed. Philadelphia, PA: WB Saunders; 2006:453–470.

11. Spagnolo V, Crippa V, Marzia A, Alberti I, Sartorelli P. Hematologic, biochemical, and protein electrophoretic values in captive tawny owls (Strix aluco). Vet Clin Pathol. 2008;37:225–228.

12. Müller K, Brunnberg L. Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye-binding method. Vet Clin Pathol. 2010;39:79–82.

13. Andreani G, Carpene E, Cannavacciuolo A, Di Girolamo N, Ferlizza E, Isani G. Reference values for hematology and plasma biochemistry variables, and protein electrophoresis of healthy Hermann's tortoises (Testudo hermanni ssp.). Vet Clin Pathol. 2014;43:573–583.

14. Flower JE, Byrd J, Cray C, Allender MC. Plasma electrophoretic profiles and hemoglobin binding protein reference intervals in the Eastern box turtle (Terrapene carolina carolina) and influences of age, sex, season, and location. J Zoo Wildl Med. 2014;45:836–842.

  

Speaker Information
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Z. Knotek
Avian and Exotic Animal Clinic
Faculty of Veterinary Medicine
University of Veterinary and Pharmaceutical Sciences Brno
Brno, Czech Republic


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