Post on 03-Apr-2018
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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
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Reptiles
Cheloniansdorsal coccygeal vein
Luer 25G x 1,5 needle
jugular vein
insulin set
Crocodilesventral coccygeal vein - Luer 18G x 1,5needle
inserted at an angle 30 - 60
then advanced at an angle 60 - 90
Lizards + Snakesventral coccygeal vein - Luer 25G x 1,5 needle
inserted at an angle 30 - 90
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Anticoagulant ...
EDTA red blood cells hemolysis
HEPARIN optimal (no risk of hemolysis)
for plasma
Na-Heparin 50 I.U./ml one drop
Li-Heparin
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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
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RBC + WBC
Natt Herrick Solution and Stain
Sodium chloride (NaCl) 3.88 g
Sodium sulfate (Na2SO4) 2.50 g
Sodium phosphate (Na2HPO4) 1.74 g
Potassium phosphate (KH2HPO4) 0.25 g
Formalin (37%) 7.5 ml
Methyl violet 0.10 g
+ to 1 000 ml with distilled water + filter
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Staining methods
Wrights Stain(for Malaria) (Lane 1991)
Wrights stain, powdered 0.30 g
Glycerol, neutral 3.00 ml
Methyl alcohol, pure 97.0 ml
Giemsa Stain(for Malaria) (Lane 1991)
Giemsa stain, powdered 0.75 g
Glycerol, pure 35.00 ml
Methyl alcohol, pure 65.00 ml
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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
Distilled water to 1.0 l
Solution B 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
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Iguana iguana
(orderSquamata, suborderSauria)
~ 40 75 % patients of the class Reptilia
~ 80 % patients suffering from nephropathy,hepatopathy, osteopathy (Brno)
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Iguana iguana
Iguanaiguana 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 ventralis1. sample (age 10 msc
2. - 6. samples (intervals of 30 days)
PCV, erythrocytes, WBC, differential counts
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Erythrocytes + PCV
RBC 0,9 - 1,9 (T/l) (1012/l)
oval shape, eosinophil. cytoplasma
intracytoplasmatic inclusions (?)
oval to round nucleus
erythroblasts (
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WBC
9,1 - 25,1 (G/l) (109/l)
granulocytes
heterophils eosinophils
basophils
lymphocytes
monocytes + azurophils (granulocyty)
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LymphocytesLymphocytes
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
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Thrombocytes
different size and shape
round to oval nucleus
cytoplasma
blue to grey PAS positive
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Heterophils (granulocytes)
0,9 - 10,2 (G/l) (109/l)
9,5 - 43,8 %
nucleus with segments
cytoplasmatic granules
rods orange to red
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Eosinophils (granulocytes)
0,2 - 1,5 (G/L) (109/l)
1,5 - 6,5 %
nucleus round to oval
cytoplasmatic granules
round blue !!!
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Basophils (granulocytes)
0,2 - 1,7 (G/l) (109/l)
3,0 - 9,0 %
nucleus round to oval
cytoplasmatic granules
round dark blue
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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
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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
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Basic plasma biochemistry profiles in reptiles
Parametr Units Crocodiles Chelonians Lizards Snakes
TP g/l 53,0 46,0 56,0 65,0
Glucose mmol/l 5,5 10,0 12,1 8,9
ALP kat/l 2,5 5,5 2,6 1,8ALT kat/l 0,6 0,7 0,8 0,5AST kat/l 1,1 1,6 1,2 0,4
Uric Acid mol/l 80,0 160,0 89,3 250,0Ca mmol/l 2,7 3,0 3,3 2,3
P mmo/l 1,0 1,5 1,6 1,3
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FEMALES
POOS POFS
MALES
Mean Mean Mean
TP g/l 59,3 56,8 49,3
Gluco mmol/l
12,7 12,3 12,7
UA mol/l
151,1 320,6 90,5
CHOL mol/l
8,2 9,1 5,1
ALT kat/l 0,4 0,3 0,5AST kat/l
0,8 0,8 0,3
Cammol/
l 2,1 6,9 2,8
Pmmol/
l 3,7 3,5 2,1
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Iguanas - renal disease Healthy iguanas
Boyer et al. (1996) Knotek et al.
(2002)
Knotek et al. (1999)
Mean Mean Mean
TP g/l 63,00 53,51 22,0 78,2
Gluco mmol/l
9,55 10,07 8,3 16,5
Uric
acid
mol/l
428,26 306,70 70,2 145,3
Creat mol/l
52,20 61,07 8,8 66,3
ALT kat/l 3,52 0,74 0,1 1,2AST kat/l
3,11 5,02 0,1 1,6K mmol
/l6,10 4,18 1,3 5,2
Ca mmol/l
1,95 2,06 2,2 3,5
P mmol
/l
7,69 5,95 1,4 3,1
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Agrionemys horsfieldi- plasma biochemistry
Wasting tortoises (n=60) Healthy tortoises (n=20)
min. max. mean min. max. mean
Total protein g/l 20.0 49.3 30.8 25.0 68.5 42.8
Glucose mmol/l 2.9 9.5 6.6 8.9 12.5 11.0
Uric acid mol/l 86.8 1 594.0 580.6 56.0 152.0 82.0ALP kat/l 3.1 12.8 5.3 2.6 8.8 4.5
ALT kat/l 0.8 3.4 1.3 0.5 1.9 0.8
AST kat/l 0.7 2.8 1.9 0.5 1.8 1.1
GMT kat/l < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Cholest. mmol/l 2.6 9.1 7.4 3.2 6.8 4.2
Na mmol/l 139.0 145.0 143.0 139.0 145.0 143.0K mmol/l 3.4 5.0 4.5 2.9 4.6 4.3
Ca mmol/l 0.8 2.2 1.0 1.9 2.8 2.5
P mmol/l 0.9 8.9 5.3 1.2 2.5 1.5
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Serum bile acid concentrationsin red-eared terrapin females
with active folliculogenesis.
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Liver diseases in chelonians
Acute ALT, AST ...
Chronic ?
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Serum concentration of BA
indirect control of liver functionduring the reproductive season
in captive red-eared terrapinfemales
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Mean serum BA concentration
8.69 5.11 mol/l
(99 % interval 6.16 11.22)
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The yolk protein precursors vitellogenins- are formed exclusively in the liver
(Hubert, 1985) under stimulation byestradiol (Ferguson, 1985).
Enlarged liver of pale colour appears tobe a normal physiological phenomenonrelated to metabolism in female reptilesduring vitellogenesis (McArthur et al.,2004).
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Determination of serum concentrations ofBA as the indirect method for monitoring
the hepatic function should be used inresearch focused on reptilian reproduction
and metabolism.
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Haematology - reptiles
Round to oval, kidney
shaped
Grey, like monocytes, but with
red-purple granulesAzurophils
Large, round to oval,
kidney shaped
Grey, pale blue to greyMonocytes
Round to oval, often is
not visible
Dark blue granulesBasophils
Lobed or unlobedGranules red, oval
Blue in some species(Iguana, Tupinambis)
Eosinophils
Unlobed, eccentric,Oval to round,
segmented
Granules bright red/ orangeoval, round, spindle.
Cytoplasma blue to grey
Heterophils
NucleusCytoplasmaCell type
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Haematology - birds
Dark blue, roundBlue to dark blue
Limited space
Lymphocytes
Large, round to oval,kidney shaped
Grey, pale blue to greyMonocytes
Round to oval, often is
not visible
Dark blue granulesBasophils
Lobed or unlobedGranules red, oval
Blue in some species
(Amazonia)
Eosinophils
Unlobed, eccentric,Oval to round,
segmented
Colourless, Granules bright red/orange
oval, round, spindle, rods
Heterophils
NucleusCytoplasmaCell type
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Variability
High in summer, sex, low in winter
Increase post hibernation
0 80Lymphocytes
High in young reptiles0 - 30Monocytes
Azurophils
High in terrestrial turtles0 40Basophils
Lowest in summer, highest during
hibernation, high in turtleslow in snakes
0 20Eosinophils
Season, sex, age,up to 40Heterophils
InfluencePortion (%)Cell type
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Changes
Inflammation, trauma, reproductionLymphocytosis
Chronic necroses, infectionsMonocytosis
Azurophilia
Suspect parasites (blood) , chronic infectionBasophilia
Inflammation, trauma, no direct correlation with
parasitesEosinophilia
Bacterial infections, inflammation, reproductionHeterophilia
Association with problemsCell type
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Birds
Blood samples collection
vena jugularis (right)
vena ulnaris
vena metatarsalis media
vena tibialis caudalis
vena brachialis
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Anticoagulant ...
EDTA only short contact with sample !
HEPARIN optimal for plasma
Na-Heparin
Li-Heparin
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Basic plasma biochemistry profiles in birds
Parametr Units Toucan Ostrich Common Crow Snowy Owl
TP g/dl 2.80 4.40 2.4 5.3 3.7 5.3
Albumin g/dl 1.4 2.4 1.1 2.3 1.8 1.4
Globulin g/dl 1.4 2.2 1.4 3.1 1.8 2.4
Glucose mg/dl 222 - 363 164 - 330 366 339.1
ALP IU/l 14 - 88 - 262 75
LDH IU/l 180 - 319 408 - 1236 291 337
AST IU/l 141 - 340 226 - 547 318 252
Uric Acid IU/l 2.4 - 14 1.0 15.0 12.1 0.5 (mol/l)
Ca mg/dl 8.8 11.8 8.0 13.6 8.2 9.4
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Basic plasma biochemistry profiles in birds
Parametr Units Amazon Parrot Cockatiel African Grey Macaw
TP g/dl 3.0 5.0 2.2 5.0 3.0 5.0 3.0 5.0
Glucose mg/dl 220 - 350 200 - 450 190 - 350 200 - 350
Uric Acid IU/l 2.0 10.0 3.5 11.0 4.0 10.0 2.5 11.5
AST IU/l 130 - 350 100 - 350 100 - 350 100 - 280
LDH IU/l 160 - 420 125 - 450 150 - 450 75 - 425
Ca mg/dl 8.0 13.0 8.5 13.0 8.0 13.0 9.0 13.0
K mg/dl 3.0 4.5 2.5 4.5 2.6 4.2 2.5 4.5
Na mg/dl 136 152 132 150 134 152 136 - 155
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Small mammalsFerret
vena cava cranialis
v. jugularis
Rat
vena saphena lateralis
vena cava cranialis
Rabbit
vena auricularis
vena saphena lateralis
Guinea pigvena cava cranialis
v. brachialis
v. saphena lateralis
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Anticoagulant ...
EDTA feasible anticoagulant
HEPARIN feasible anticoagulant
Na-Heparin
Li-Heparin
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Laboratory small mammals
PCV centrifuge
Haemoglobin concentrations by cyanmethemoglobin method
RBC and WBC performed manually, using a hemocytometer with
Trk and Hayem stain solutions
automat cell counter
Blood smears Diff-Quick
Pappenheim method of biphasic staining
May-Grnwald and Giemsa-Romanowski
Wrights Giemsa
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RBC + WBC
Hayems Stain (RBC)
HgCl2 0.25 g
NaCl 0.50 g
Na2SO4 2.50 gDistilled water to 100.0 g
Trks Stain (WBC)CH3COOH 1 ml
Methyl violet 1% water sol. 1 ml
Distilled water to 100.0 ml
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Staining methods
May-Grnwald sol.
eosine + methylene blue 1.00 g
Methyl alcohol 100.0 g
Glycerol 50.0 g
Giemsa-Romanowski sol.azureosin II 3.00 g
azur II 0.80 g
Glycerol 250.0 g
Methyl alcohol 0.35 g
Diluted before use (1 + 9 with H2O)
Distilled water (buffered)
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Basic plasma biochemistry profiles in mammals
Parametr Units Ferret Guinea pig Rabbit Rat
TP g/l 53.0 72.0 46.0 62.0 54.0 75.0 56.0 76.0
Glucose mmol/l 5.2 11.4 3.3 6.9 4.1 8.7 2.8 7.5
ALP IU/l 30.0 120.0 66.0 74.0 < 120 70.0 132.0
ALT IU/l 30.0 100.0 39.0 45.0 < 100 26.0 37.0AST IU/l 15.0 40.0 46.0 48.0 < 100 40.0 53.0
Creatinin mol/l 35.5 79.5 53 - 194 70.7 159.0 17.5 70.8BUN mmol/l 1.7 7.5 1.5 5.2 2.8 3.9 0.8 3.5
Bilirubin mmo/l 4.0 - 17.1 5.1 15.4 4.3 12.6 3.4 10.3
Ca mmo/l 2.0 2.9 1.3 2.8 1.4 3.1 2.6 3.2
P mmo/l 1.3 2.9 1.0 2.4 1.3 1.9 1.7 2.7Na mmo/l 137 - 162 152 - 162 138 - 155 143 156
K mmo/l 4.5 7.7 6.8 8.9 3.7 6.8 5.4 6.4
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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 ProfessionalPublishing Ltd., 2002:410.
Knotkova Z, Doubek J, Knotek Z, Hajkova P Blood Cell Morphology and Plasma Biochemistry in RussianTortoises (Agrionemys horsfieldi). Acta Veterinaria Brno, 2002, 71:191 - 198
Knotek Z, Hauptman K, Knotkova Z, Hajkova P, Tichy F Haemogram and Plasma Biochemistry in GreenIguanas with Renal Disease. Acta Veterinaria Brno, 2002, 71:333 - 340
Knotek Z, Knotkova Z, Doubek J, Pejrilova S, Hauptman K Plasma Biochemistry in Female GreenIguanas (Iguana iguana) with Calcium Metabolism Disorders. Acta Veterinaria Brno, 2003, 72:183 189
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Pejrilova S, Knotkova Z, Knotek Z, Vrbas J Age-Related Changes of the HaematologicalProfile 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 theNorway rat (Rattus norvegicus). Laboratory Animals 2005, 39:236 239
Knotkova Z, Mazanek S, Hovorka M, Sloboda M, Knotek Z Blood profile of Bornean river turtlessuffering 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 ReproductiveSeason upon Plasma Biochemistry Values in Green Iguanas Acta Vet. Brno, 2005, 74: 515-520
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