Haematology and clinical biochemistry of exotic animals

univ. prof. Zdenek Knotek, DVM PhD. Zora Knotkova, DVM, PhD.
Univ. of Veterinary and Pharm. Sci. Brno Czech Republic

Reptiles
Chelonians
dorsal coccygeal vein Luer 25G x 1,5 needle jugular vein insulin set

Crocodiles
ventral coccygeal vein - Luer 18G x 1,5 needle inserted at an angle 30 - 60 then advanced at an angle 60 - 90

Lizards + Snakes
ventral coccygeal vein - Luer 25G x 1,5 needle inserted at an angle 30 - 90

Anticoagulant ...
EDTA red blood cells hemolysis

HEPARIN optimal (no risk of hemolysis) for plasma Na-Heparin Li-Heparin 50 I.U./ml one drop

Laboratory reptiles and birds

PCV

centrifuge

Haemoglobin concentrations by cyanmethemoglobin method RBC and WBC performed manually, using a hemocytometer with Natt and Herrick stain (solution 1:100 dilution) Blood smears Diff-Quick Pappenheim method of biphasic staining May-Grnwald and Giemsa-Romanowski Wrights Giemsa

RBC + WBC
Natt Herrick Solution and Stain
Sodium chloride Sodium sulfate Sodium phosphate Potassium phosphate Formalin (37%) Methyl violet (NaCl) (Na2SO4) (Na2HPO4) (KH2HPO4) 3.88 g 2.50 g 1.74 g 0.25 g 7.5 ml 0.10 g

+ to 1 000 ml with distilled water + filter

Staining methods
Wrights Stain (for Malaria)
Wrights stain, powdered Glycerol, neutral Methyl alcohol, pure

(Lane 1991)
0.30 g 3.00 ml 97.0 ml

Giemsa Stain (for Malaria)

Giemsa stain, powdered Glycerol, pure Methyl alcohol, pure 0.75 g 35.00 ml 65.00 ml

(Lane 1991)

Staining methods
pH of the buffered water (Lane 1991)
distilled water pH = 5.5 tap water pH = 6.0 optimal buffered water pH = 7.2 (7.0 7.5) Solution A Disodium Phosphate (Sigma # S 0876) (Na2 HPO4) 9.50 g Solution B Distilled water to 1.0 l Monosodium Phosphate (Sigma # S 0751) (NaH2PO4 . 2 H2O) 9.20 g

Distilled water to 1.0 l optimal buffered water pH = 7.2 A (72.0 ml) + B (28.0 ml) + distilled water 900.0 ml

Iguana iguana (order Squamata, suborder Sauria) ~ 40 75 % patients of the class Reptilia ~ 80 % patients suffering from nephropathy, hepatopathy, osteopathy (Brno)

Iguana iguana
Iguana iguana n = 12 8 males, 4 females 4 breeding goups (1,0 + 1,0 + 0,1) air temperature 24 - 35 C air humidity 60 - 80 % light-regime 12 h/12 h samples v. coccygea ventralis 1. sample (age 10 msc 2. - 6. samples (intervals of 30 days) PCV, erythrocytes, WBC, differential counts

Erythrocytes + PCV

RBC 0,9 - 1,9 (T/l) (1012/l) oval shape, eosinophil. cytoplasma intracytoplasmatic inclusions (?) oval to round nucleus erythroblasts (<1 %) PCV 0,35 - 0,45 (l/l)

WBC 9,1 - 25,1 (G/l) (109/l) granulocytes heterophils eosinophils basophils lymphocytes monocytes + azurophils (granulocyty)

Lymphocytes

4,8 - 14,0 (G/l) (109/l) 36,8 - 81,0 % the biggest subpopulation of leucocytes round nucleus blue cytoplasma lymphocytes x thrombocytes PAS negative

Thrombocytes

different size and shape round to oval nucleus cytoplasma blue to grey PAS positive

Heterophils (granulocytes)

0,9 - 10,2 (G/l) (109/l) 9,5 - 43,8 % nucleus with segments cytoplasmatic granules rods orange to red

Eosinophils (granulocytes)

0,2 - 1,5 (G/L) (109/l) 1,5 - 6,5 % nucleus round to oval cytoplasmatic granules round blue !!!

Basophils (granulocytes)

0,2 - 1,7 (G/l) (109/l) 3,0 - 9,0 % nucleus round to oval cytoplasmatic granules round dark blue

Monocytes

0,2 - 1,7 (G/l) (109/l) 1,2 - 7,2 % cells of different size nucleus oval to lobates cytoplasma grey-blue with vacuoles

Azurophils (granulocytes)

0,1 - 4,9 (G/l) (109/l) 1,8 - 12,2 % nucleus oval to round situated lateraly to the center cytoplasmatic granules round azurophilic

Basic plasma biochemistry profiles in reptiles

Parametr TP Glucose ALP ALT AST Uric Acid Ca P Units g/l mmol/l kat/l kat/l kat/l mol/l mmol/l mmo/l Crocodiles 53,0 5,5 2,5 0,6 1,1 80,0 2,7 1,0 Chelonians 46,0 10,0 5,5 0,7 1,6 160,0 3,0 1,5 Lizards 56,0 12,1 2,6 0,8 1,2 89,3 3,3 1,6 Snakes 65,0 8,9 1,8 0,5 0,4 250,0 2,3 1,3

FEMALES POOS
Mean

MALES
Mean Mean

POFS 56,8 12,3 320,6 9,1 0,3 0,8 49,3 12,7 90,5 5,1 0,5 0,3

TP Gluco UA CHOL ALT AST

g/l
mmol/ l

59,3 12,7 151,1 8,2 0,4 0,8

mol/l mol/l kat/l kat/l

Ca P

mmol/ l mmol/ l

2,1 3,7

6,9 3,5

2,8 2,1

Iguanas - renal disease

Boyer et al. (1996) Knotek et al. (2002)

Healthy iguanas
Knotek et al. (1999)

Mean TP Gluco Uric acid Creat ALT AST K Ca P

g/l mmol /l mol/l mol/l kat/l kat/l mmol /l mmol /l mmol /l

Mean

Mean

63,00 9,55 428,26 52,20 3,52 3,11 6,10 1,95 7,69

53,51 10,07 306,70 61,07 0,74 5,02 4,18 2,06 5,95

22,0 78,2 8,3 16,5 70,2 145,3 8,8 66,3 0,1 1,2 0,1 1,6 1,3 5,2 2,2 3,5 1,4 3,1

Agrionemys horsfieldi - plasma biochemistry

Wasting tortoises (n=60) min. max. mean 20.0 49.3 30.8 2.9 9.5 6.6 86.8 1 594.0 580.6 3.1 12.8 5.3 0.8 3.4 1.3 0.7 2.8 1.9 < 0.1 < 0.1 < 0.1 2.6 9.1 7.4 139.0 145.0 143.0 3.4 5.0 4.5 0.8 2.2 1.0 0.9 8.9 5.3 Healthy tortoises (n=20) min. max. mean 25.0 68.5 42.8 8.9 12.5 11.0 56.0 152.0 82.0 2.6 8.8 4.5 0.5 1.9 0.8 0.5 1.8 1.1 < 0.1 < 0.1 < 0.1 3.2 6.8 4.2 139.0 145.0 143.0 2.9 4.6 4.3 1.9 2.8 2.5 1.2 2.5 1.5

Total protein g/l Glucose mmol/l Uric acid mol/l ALP kat/l ALT kat/l AST kat/l GMT kat/l Cholest. mmol/l Na mmol/l K mmol/l Ca mmol/l P mmol/l

Serum bile acid concentrations in red-eared terrapin females with active folliculogenesis.

Liver diseases in chelonians Acute ALT, AST ...

Chronic ?

Serum concentration of BA indirect control of liver function during the reproductive season in captive red-eared terrapin females

Mean serum BA concentration 8.69 5.11 mol/l (99 % interval 6.16 11.22)

The yolk protein precursors vitellogenins - are formed exclusively in the liver (Hubert, 1985) under stimulation by estradiol (Ferguson, 1985). Enlarged liver of pale colour appears to be a normal physiological phenomenon related to metabolism in female reptiles during vitellogenesis (McArthur et al., 2004).

Determination of serum concentrations of BA as the indirect method for monitoring the hepatic function should be used in research focused on reptilian reproduction and metabolism.

Haematology - reptiles
Cell type Cytoplasma
Granules bright red/ orange oval, round, spindle. Cytoplasma blue to grey Granules red, oval Blue in some species (Iguana, Tupinambis) Dark blue granules

Nucleus
Unlobed, eccentric, Oval to round, segmented Lobed or unlobed

Heterophils Eosinophils Basophils Monocytes Azurophils

Round to oval, often is not visible Large, round to oval, kidney shaped Round to oval, kidney shaped

Grey, pale blue to grey

Grey, like monocytes, but with red-purple granules

Haematology - birds
Cell type Cytoplasma Nucleus

Heterophils Eosinophils Basophils Monocytes

Lymphocytes

Colourless, Granules bright red/ Unlobed, eccentric, orange Oval to round, oval, round, spindle, rods segmented Granules red, oval Blue in some species (Amazonia) Dark blue granules Lobed or unlobed

Round to oval, often is not visible Large, round to oval, kidney shaped Dark blue, round

Grey, pale blue to grey

Blue to dark blue Limited space

Variability
Cell type Portion (%)
up to 40

Influence
Season, sex, age,

Heterophils Eosinophils Basophils Monocytes Azurophils

Lymphocytes

0 20

Lowest in summer, highest during hibernation, high in turtles low in snakes High in terrestrial turtles High in young reptiles

0 40 0 - 30

0 80

High in summer, sex, low in winter Increase post hibernation

Changes

Cell type

Association with problems

Bacterial infections, inflammation, reproduction Inflammation, trauma, no direct correlation with parasites Suspect parasites (blood) , chronic infection Chronic necroses, infections

Heterophilia Eosinophilia Basophilia Monocytosis Azurophilia Lymphocytosis

Inflammation, trauma, reproduction

Birds Blood samples collection

vena jugularis (right) vena ulnaris vena metatarsalis media vena tibialis caudalis vena brachialis

Anticoagulant ...
EDTA only short contact with sample !

HEPARIN Na-Heparin Li-Heparin

optimal for plasma

Basic plasma biochemistry profiles in birds

Parametr Units Toucan 2.80 4.40 1.4 2.4 1.4 2.2 222 - 363 14 - 88 180 - 319 141 - 340 2.4 - 14 8.8 11.8 Ostrich 2.4 5.3 1.1 2.3 1.4 3.1 164 - 330 408 - 1236 226 - 547 1.0 15.0 8.0 13.6 Common Crow 3.7 1.8 1.8 366 262 291 318 12.1 8.2 Snowy Owl 5.3 1.4 2.4 339.1 75 337 252 0.5 (mol/l) 9.4

TP g/dl Albumin g/dl Globulin g/dl Glucose mg/dl ALP IU/l LDH IU/l AST IU/l Uric Acid IU/l Ca mg/dl

Basic plasma biochemistry profiles in birds

Parametr TP Glucose Uric Acid AST LDH Ca K Na Units Amazon Parrot g/dl mg/dl IU/l IU/l IU/l mg/dl mg/dl mg/dl 3.0 5.0 220 - 350 2.0 10.0 130 - 350 160 - 420 8.0 13.0 3.0 4.5 136 152 Cockatiel 2.2 5.0 200 - 450 3.5 11.0 100 - 350 125 - 450 8.5 13.0 2.5 4.5 132 150 African Grey 3.0 5.0 190 - 350 4.0 10.0 100 - 350 150 - 450 8.0 13.0 2.6 4.2 134 152 Macaw 3.0 5.0 200 - 350 2.5 11.5 100 - 280 75 - 425 9.0 13.0 2.5 4.5 136 - 155

Small mammals
Ferret vena cava cranialis v. jugularis Rat vena saphena lateralis vena cava cranialis Rabbit vena auricularis vena saphena lateralis Guinea pig vena cava cranialis v. brachialis v. saphena lateralis

Anticoagulant ...
EDTA feasible anticoagulant

HEPARIN Na-Heparin Li-Heparin

feasible anticoagulant

Laboratory small mammals

PCV

centrifuge

Haemoglobin concentrations by cyanmethemoglobin method RBC and WBC performed manually, using a hemocytometer Trk and Hayem stain solutions automat cell counter Blood smears Diff-Quick Pappenheim method of biphasic staining May-Grnwald and Giemsa-Romanowski Wrights Giemsa with

RBC + WBC
Hayems Stain
HgCl2 NaCl Na2SO4 Distilled water to (WBC) 1 ml 1 ml 100.0 ml (RBC) 0.25 g 0.50 g 2.50 g 100.0 g

Trks Stain
CH3COOH

Methyl violet 1% water sol. Distilled water to

Staining methods
May-Grnwald sol.
eosine + methylene blue Methyl alcohol Glycerol 1.00 g 100.0 g 50.0 g 3.00 g 0.80 g 250.0 g 0.35 g

Giemsa-Romanowski sol.
azureosin II azur II Glycerol Methyl alcohol

Diluted before use (1 + 9 with H2O)

Distilled water (buffered)

Basic plasma biochemistry profiles in mammals

Parametr TP Glucose ALP ALT AST Creatinin BUN Bilirubin Ca P Na K Units g/l mmol/l IU/l IU/l IU/l mol/l mmol/l mmo/l mmo/l mmo/l mmo/l mmo/l Ferret 53.0 72.0 5.2 11.4 30.0 120.0 30.0 100.0 15.0 40.0 35.5 79.5 1.7 7.5 4.0 - 17.1 2.0 2.9 1.3 2.9 137 - 162 4.5 7.7 Guinea pig 46.0 62.0 3.3 6.9 66.0 74.0 39.0 45.0 46.0 48.0 53 - 194 1.5 5.2 5.1 15.4 1.3 2.8 1.0 2.4 152 - 162 6.8 8.9 Rabbit 54.0 75.0 4.1 8.7 < 120 < 100 < 100 70.7 159.0 2.8 3.9 4.3 12.6 1.4 3.1 1.3 1.9 138 - 155 3.7 6.8 Rat 56.0 76.0 2.8 7.5 70.0 132.0 26.0 37.0 40.0 53.0 17.5 70.8 0.8 3.5 3.4 10.3 2.6 3.2 1.7 2.7 143 156 5.4 6.4

Literature
Beynon PH, Lawton, MPC, Cooper, JE (Eds.) Manual of Reptiles. BSAVA, Cheltenham, 1992. Meredith A, Redrobe S (Eds.) BSAVA Manual of Exotic Pets. Quedgeley, 2002. Fowler ME, Miller RE (Eds.) Zoo and Wild Animal Medicine. Saunders, St. Louis, 2003. Jacobson ER (Ed.) Biology, Husbandry, and Medicine of the Green Iguana. Krieger Publ. Comp., Maabar, 2003. Flecknell P (Ed.) Manual of Rabbit Medicine and Surgery. Gloucester; BSAVA, 2000. Harkness JE, Wagner JE (Eds.) The Biology and Medicine of Rabbit and Rodents. 4th ed., Baltimore; Williams & Wilkins, 1995. Quesenberry KE, Carpenter JW (Eds.) Ferrets, Rabbits and Rodents. Clinical Medicine and Surgery. 2nd ed. St. Louis; W.B. Saunders, 2003. Harcourt-Brown F (Ed.) Textbook of Rabbit Medicine. Oxford; Reed Educational and Professional Publishing Ltd., 2002:410. Knotkova Z, Doubek J, Knotek Z, Hajkova P Blood Cell Morphology and Plasma Biochemistry in Russian Tortoises (Agrionemys horsfieldi). Acta Veterinaria Brno, 2002, 71:191 - 198 Knotek Z, Hauptman K, Knotkova Z, Hajkova P, Tichy F Haemogram and Plasma Biochemistry in Green Iguanas with Renal Disease. Acta Veterinaria Brno, 2002, 71:333 - 340 Knotek Z, Knotkova Z, Doubek J, Pejrilova S, Hauptman K Plasma Biochemistry in Female Green Iguanas (Iguana iguana) with Calcium Metabolism Disorders. Acta Veterinaria Brno, 2003, 72:183 189

Pejrilova S, Knotkova Z, Knotek Z, Vrbas J Age-Related Changes of the Haematological Profile in Green Iguana (Iguana iguana rhinolopha). Acta Veterinaria Brno, 2004, 73: 305 31 Jekl V, Hauptman K, Jeklova E, Knotek Z Blood sampling from the cranial vena cava in the Norway rat (Rattus norvegicus). Laboratory Animals 2005, 39:236 239 Knotkova Z, Mazanek S, Hovorka M, Sloboda M, Knotek Z Blood profile of Bornean river turtles suffering from shell necrosis and haemogregarine parasites Vet. Med. Czech., 2005, 50: 421 426 Knotkova Z, Pejrilova S, Trnkova S, Matouskova O, Knotek Z Influence of Reproductive Season upon Plasma Biochemistry Values in Green Iguanas Acta Vet. Brno, 2005, 74: 515520

prof. Zdenek Knotek, DVM, PhD. Avian and Exotic Animal Clinic Faculty of Veterinary Medicine Univ. Vet. and Pharm. Sci. Brno Palackeho 1-4 CZ-612 42 Brno Czech Republic +420 5 4156 2381 knotekz@vfu.cz