Clinical examination

Normal physical parameters and basic examination tech­niques are detailed by Blanshard and Bodley (2008). The signs of disease or trauma in koalas may be overt or subtle, with some clinical signs only becoming apparent late in the disease process (e.g.

2.1 Clinical pathology

Examination of blood films, bone marrow smears, abdominocentesis smears and urinalysis (including sedi­ment cytology) are important components in the diag­nostic work-up of koalas. All can be collected safely and cost-effectively under chemical restraint, requiring rela­tively little experience and a basic understanding to effec­tively interpret findings.

2.1.1 Haematology and biochemistry

RBC morphology and biochemistry are discussed in Blanshard and Bodley (2008). Haematological and bio­chemical reference values are presented in Appendix 1.

Common haematological abnormalities include:

Table 33.1. Diagnostic tests indicated in addition to thorough physical examination for sick and injured koalas (Phascolarctos cinereus)

*Include for sick managed koala if indicated by clinical signs.

• stress leucogram - often seen in rescued free-ranging koalas brought into managed care and/or with chronic disease

• anaemia - acute blood loss through trauma, chronic blood loss from cystitis and/or reduced erythroid pro­duction associated with chronic inflammatory disease (poorly regenerative); seasonal blood loss associated with heavy tick and mosquito parasitism (regenera­tive). If blood loss is chronic and severe, iron deficiency anaemia may result, leading to a poor regenerative response

• leucocytosis - ± band neutrophils, associated with acute and/or active inflammation

• leucopenia - associated with myelodysplastic syn­dromes and excessive demand/depletion associated with severe infections

• free and/or phagocytosed bacteria associated with severe infections and sepsis

• vacuolated monocytes - associated with chronic inflammatory diseases, such as chlamydiosis

• leukaemia - most commonly of the lymphoid series, often with concurrent anaemia associated with myelophthisis

• mild elevations in some WBCs, including eosinophils, often without any explanatory clinical findings.

Myelodysplasia may lead to more subtle changes, such as mild to moderate anaemia, leucopenia, thrombocyto­penia and sometimes large and unusually-shaped WBCs, whose lineage may be difficult to determine. A diagnosis of myelodysplasia can be supported by examination of a bone marrow smear, which may show an absence or reduction of the usual clusters of erythroid and myeloid differentiation, and in some cases a background of atypi­cal blast cells.

Hypoproteinaemia is common in critically ill koalas and those with significant blood loss or septicaemia. Hypoproteinaemia may lead to peripheral, caecal wall or pulmonary oedema. TPP <45 g/L warrants intervention such as plasma transfusion (see section 3.1.1). Serum pro­tein electrophoresis analyses of zoo-based and free-ranging koalas indicate that zoo-based koalas have lower albumin and β-globulin fractions as compared with free-ranging koalas, possibly as a consequence of fewer inflammatory or infectious stimuli in managed settings (Pye et al.

2.1.2 Bone marrow examination

Bone marrow cytology is frequently indicated, given the relatively common occurrence of leukaemia and other bone marrow diseases, particularly in northern koalas. The technique for bone marrow sample collection is described in Blanshard and Bodley (2008). The two-slide pull-apart method should be used to prepare smears with only very gentle pressure. Three key features for assessing bone marrow cytology are as follows.

1. Overall nucleated cellularity - marked hypercellularity is associated with replacement of bone marrow with malignant or atypical cells in cases of leukaemia (Plate 33.1) and myelodysplasia (Plate 33.2). These abnormal cells are very fragile and often rupture, resulting in ‘smudge’ or ‘basket cells’. Care should be taken not to misinterpret erythroid or myeloid series responsive hyperplasia as leukaemia. Hypercellular bone marrow that is responding to increased demand shows normal differentiation and maturation of the blood cells, but large, immature blast cells may be more prominent. Myelophthisic marrow will be dominated by atypical and usually very fragile cells, with little or no evidence of normal differentiation and maturation. Hypocellularity may result from excessive suction when aspirating the sample, resulting in dilution of the sample with blood, or may be seen with hypocellular myelodysplastic syndromes such as aplastic anaemia.

2. Presence of all lineages of haemopoietic cells

- erythroid, myeloid and megakaryocytic cell lines should be easily identifiable in a normal bone marrow smear (Plate 33.3). Erythroid-lineage immature cells are nucleated and tend to have blue-purple cytoplasm and large nuclei. Normal clusters of erythroid differentiation also often contain mature erythroid cells that appear as nucleated RBCs. Myeloid series cells have more abundant and pinker cytoplasm, and clusters of normal differentiation often have cells resembling the granulocytic cell lines. Megakaryocytes are clearly visible in normal bone marrow as large cells with multinucleated or multilobulated nuclei that are less common but easily identified in smears.

3. Orderly differentiation and maturation of erythroid and myeloid series cells - correctly prepared smears of normal marrow should demonstrate clearly identifiable clusters of erythroid and myeloid differentiation (Plate 33.4). These clusters often feature a group of cells at varying stages of maturation from relatively undifferentiated blast cells through to easily identified mature cells of the erythroid or granulocyte lineage. In contrast, myelodysplastic marrow demonstrates no or poor differentiation and maturation, a background of atypical undifferentiated blast cells or simply an absence of normal haematopoietic cells (aplastic anaemia).

2.1.3 Abdominocentesis

Abdominocentesis and cytological examination of the fluid is an important component in the work-up of sick

Fig. 33.1. Abdominocentesis. A 25G, 25-mm winged infusion set with a 3-mL syringe attached inserted through the skin at 45-90° to approximately one-third of the depth of the needle. Negative pressure is gently applied and the needle withdrawn to obtain a peritoneal fluid sample.

and injured koalas. An alternative method for abdomi- nocentesis to that described by Blanshard and Bodley (2008) is as follows: the sedated or anaesthetised koala is positioned in lateral recumbency (either side) and the fur in the upper region of the paralumbar fossa is parted or shaved before aseptic preparation of the site. A 25G, 25-mm winged infusion set or standard hypodermic needle with a 3 mL syringe attached is inserted through the skin (at 45-90°) to approximately one-third of the depth of the needle. Negative pressure is gently applied and the needle withdrawn (Fig. 33.1). Elevating the pelvis can aid sample collection. Normal peritoneal fluid is clear and low volume. Discolouration and/or turbidity or volumes greater than 0.25 mL are abnor­mal. Contamination with, or aspiration of, caeco-colic content is common, particularly if the needle is advanced too deeply.

2.1.4 Cystocentesis

Ultrasound-guided Cystocentesis is performed using a 3-mL syringe and 25G, 38-mm needle in an anaesthetised koala placed in dorsal recumbency. The ultrasound probe is placed just cranial to the pubis in a sagittal or trans­verse plane. The needle is advanced between the probe head and the pubis into the bladder, ensuring that it is maintained within the plane of the sonographic image. On entering the bladder, a 2-mL sample is aspirated and the suction stopped before withdrawing the needle. Allowing suction to continue during withdrawal of the needle can result in aspiration of peritoneal and hindgut contents, confusing the cytological findings. Urine should be centrifuged and the sediment pellet collected and smeared on a glass slide with a small-tipped swab (such as the Copan® 160C rayon-tipped sterile dry swab, Copan Diagnostics Inc., CA, USA) before drying and staining for microscopic examination.

Normal koala urine may contain scant urinary crys­tals and spermatozoa in males. The presence of neutro­phils in urine sediment (collected by cystocentesis only) confirms cystitis and is most commonly associated with chlamydial infection. Voided or samples collected via catheter may contain neutrophils derived from other sites, such as the urethra, prostate and urogenital sinus. The presence of RBCs suggests bladder mucosal ulcera­tion, fungal/candidal elements confirm fungal cystitis (Plates 33.12 and 33.13), whereas bacteria (Plate 33.14), urinary casts and urinary crystals (Plates 33.14 and 33.15) suggests non-chlamydial (or complicated chlamydial) cystitis or renal disease such as oxalate nephrosis.

3.