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The chapter describes diagnostic techniques adapted by avian veterinarians encompassing the procedure, pros and cons of anamnesis, physical examination, collection and processing of clinical specimens, laboratory examination, radiography, USG, CT, MRI, myelography, echocardiography, electrocardiography and endoscopy.
KeywordsSterile Normal Saline Blood Biochemical Parameter Hepatic Lipidosis Avian Image Laboratory Test Report
Most of the pet bird owners prefer to visit their veterinarians during crisis period when possibility of recovery becomes low. Instead, visit to veterinarians in a regular interval, recently termed as ‘wellness examination’ will provide a knowledge package consisting of infection if present, balanced diet, proper exercise, behaviour and husbandry practices to be followed. Inexperienced bird owners and owners of aged birds with chronic ailment should have regular wellness examination of their pets. To provide this knowledge package the veterinarian should also follow appropriate diagnostic approaches with proper medicinal, nutritional, and husbandry recommendations.
Taking history from the owner regarding health, diet and husbandry practices of their pet birds is the most primitive but still considered as most valuable diagnostic technique. This is a two-way technique which depends on co-operation of the owners, questioning ability and patience of the veterinarians and moreover, proper record keeping. The veterinarian can know whether the ailment is new or progressed from earlier stage. Co-operation from owners with truthful replies is required for proper diagnosis.
The veterinarians should primarily focus on age, species, sex, physical activities, urination and droppings (colour, consistency and amount) of the birds. Exposure to environment such as soil, garden, toxic fume or smoke, bird shows/fairs, shelters, aviaries, other infected birds and wild birds should be enquired from the owners. Evaluation of earlier recommendations regarding diet, bedding materials is necessary. What exactly the bird is taking as feed, not what is provided to them is important to know. Excess or deficiency of nutrients is associated with different diseases. Excess fat in diet (present in seeds) causes obesity and hepatic lipidosis. Deficiency of vitamin and calcium causes osteoporosis and impairment of vision. The veterinarian can judge the possibility and severity of a specific ailment depending on the species, age and exposure of the birds. Sub-clinical carriers of zoonotic infections should also be identified specially when the birds are reared by aged or immunosuppressed owners. To perform all these functions properly the veterinarians should have an updated knowledge other than experience.
5.2 Physical Examination
Physical examination of the bird should be performed in a clean perch or small enclosure. Gentle restraining of the birds is necessary not only to avoid injury of the birds but also to gain the trust of the owners. Bad handling always creates doubt about the ability of a veterinarian. Mild sedation with midazolam (0.2–0.5 mg/kg body weight, intramuscular) or butorphanol (1–2 mg/kg body weight, intramuscular) will help to restrain hyperactive birds.
5.3 Collection of Clinical Samples
Collection of blood samples from different avian veins
Blood collection sites
Right jugular vein
Flamingos, budgerigars, raptors, penguins, ostrich
Unlike mammals, avian jugular vein is not located in any furrow. It is present subcutaneously at right neck. Due to fat deposition in neck, jugular vein is unsuitable for blood collection from pigeons
Most of the birds (for e.g. snow goose, gull, bald ibis, pigeon, bald eagle, Eurasian kestrel, hawk, thrush, crane, African grey parrot, rhea etc.)
Possibility of haematoma formation is more in small birds and racing pigeons
Medial metatarsal vein
Pigeons, raptors, ducks
Possibility of haematoma formation is low
After cleaning the claws with antiseptic it is cut and the blood is collected in capillary tubes. The bleeding can be stopped with silver nitrate or ferrous subsulphate
Collection of avian serum in sufficient volume to conduct all the required tests is difficult due to presence of large fibrin clots which often decreases the volume. Heparinized plasma or whole blood is most common sample available from avian practitioners.
5.3.2 Droppings/Cloacal Swabs
Fresh droppings or faeces should be collected in sterile, screw capped short jars or plastic vials. Rubber caps should be avoided because the gas generated in the collected faeces may blow the cap. The faeces and urine should not be mixed together.
5.3.3 Crop/Proventriculus Washing
Normal saline (warm, 20 ml/kg body weight) is instilled into the crop or proventriculus with a blunt ended catheter or gavage tube. A part of the fluid is aspirated out with gentle negative pressure. For proventriculus washing, endotracheal tube can be used with general anaesthesia.
5.3.4 Air Sac Washing
A sterile catheter attached with a syringe is inserted into the last intercostal space of a bird and sterile normal saline (3 ml for large birds) is injected and aspirated out immediately.
5.3.5 Tracheal Washing
Nylon tube (1 mm in diameter) or a canine catheter is inserted through the glottis and sterile normal saline (0.5–2 ml/kg body weight) is flushed and the washing is collected immediately. It is safe to collect tracheal washing from highly sedated birds.
5.3.6 Autopsy Samples from Vital Organs
The incised portion of the vital organ (liver) is wiped gently with a paper towel to remove excess blood. Indurated tissue is scraped with a sterile scalpel and the tissue is kept over the slide for further examination.
5.3.7 Bone Marrow
Bone marrow is collected from proximal tibiotarsal bone or keel of the sternum. Paediatric bone biopsy needle (23 s.w.g.) can be used for bone marrow collection. Bone marrow samples are useful for confirmation of leukaemia and non-regenerative anaemia.
5.4 Processing of Clinical Samples
Blood, exudates or aspirates from the lesions are required to mix with EDTA (or heparin) to prevent clotting. Physical parameters of the fluid samples such as colour (haemolysis), specific gravity, turbidity (presence of fat particles) are judged. Cellular part of the fluid is concentrated by low speed centrifugation (1500 rpm for 10 min).
5.5 Diagnostic Techniques Used in Laboratory
A smear can be prepared with the fluid sample for detection of microbes and parasites, morphology and differential counts of cellular components (heterophils, macrophages, leukocytes) and cells with malignancy.
Standard values of blood cells, biochemical parameters, enzymes, electrolytes in birds
Packed cell volume (PCV)
Young birds (not fully fledged) have lower PCV
Decreased haemoglobin and PCV (haemolytic anaemia): blood parasite infection (Haemoproteus, Plasmodium, Leucocytozoon); gastrointestinal parasitism (Capillaria, ascarids, coccidiosis, giardiasis); mite infestation; bacterial and yeast infection (salmonellosis, colibacillosis, yersiniosis, Macrorhabdus , proventricular ulceration, campylobacteriosis); viral infection (Pacheco’s virus, Herpes virus)
Mean corpuscular volume (MCV)
121–200 fl (psittacines: 99 fl; cassowary: 280 fl)
Increased value indicates regenerative or macrocytic anaemia
Mean corpuscular haemoglobin concentration (MCHC)
Reduced MCHC index (Chronic non-regenerative anaemia): chronic infection (chlamydophilosis, toxoplasmosis, aspergillosis, salmonellosis, yersiniosis, colibacteriosis, campylobacteriosis); toxicosis [lead, copper, zinc, chloramphenicol, pesticides (DDT, carbamates), aflatoxins]; starvation and malnutrition
Raptors: above 3.5%
Total erythrocyte count (TEC)
2.1–5.5 × 1012/l
(Mean: 3.9 × 1012/l)
Young birds (not fully fledged) have lower TEC; Higher in migratory birds during flying
1–32 × 109/l
[heterophils 20–75%, lymphocytes 20–65%, monocytes 2–5%, basophils 2.5–6%, eosinophils 1–4%]
Leucocytosis-infection with bacteria, fungi, parasite, neoplasia, trauma
Heteropenia, lymphopenia- Viral infection
Thrombocyte count (platelet)
20–30 × 109/l
Total serum protein
Hypoproteinaemia—Hepatopathy, gastrointestinal parasitism, nephritis, trauma, lead toxicosis, Pacheco’s disease, anaemia, malnutrition etc.
Hyperproteinaemia—prior to egg laying stage in physiological condition, dehydration, acute infection and shock
Total plasma protein
0.15 g/dl above the value of serum protein
Albumin/globulin ratio (A/G)
Decreased A/G ratio—acute and chronic infections (e.g. Chlamydophila, aspergillosis, mycobacteriosis)
200–500 mg/dl (Emu: 158 mg/dl)
Normal value varies with age, diet, breeding season of the birds. The serum glucose level is decreased during day time and increases during night (reverse for nocturnal birds)
Hyperglycaemia—stress, lead toxicosis, pancreatitis, Diabetes mellitus in granivorous birds, budgerigars, cockatoos, Amazon parrots, macaws, cockatiels, toucans
Hypoglycaemia—starvation, hypovitaminosis, septicaemia
Normal value is more in carnivorous birds than the granivorous birds.
High level of uric acid—starvation, gout, trauma, toxicity (due to excess gentamicin, sulphonamides, azole group antifungals), hypervitaminosis D3, bacterial or viral infection
High level of plasma urea-dehydration, cardiopathy, cloacal impaction, neoplasm, blockage of renal tubules with urate crystals during salt poisoning
High level—fatty degeneration of liver, xanthomatosis (yellowish cholesterol deposition in any tissue)
[Most of the species produce biliverdin, not bilirubin. True jaundice (icterus) is not detected in birds (except in macaws when bilirubin concentration is >2.36 mg/dl)]
18–144 µmol/l (psittacines)
High bile acid level in plasma—hepatopathy
Aspartate amino transferase (AST)
High level indicates hepatopathy, Pacheco’s disease, chlamydophilosis, toxicosis due to pesticides, adverse reaction of drugs (doxycycline injection, azole group of antifungals)
Alanine amino transferase (ALT)
Lactate dehydrogenase (LDH)
Alkaline phosphatase (ALP)
High ALP level—osteomyelitis, bone neoplasms, fractures, aflatoxin poisoning, rickets, hyperparathyroidism, physiologically high during egg laying period
Creatine phosphokinase (CPK)
High CPK level—convulsions, lead toxicity, chlamydophilosis, bacterial septicaemia, vitamin E deficiency
(Budgerigars: 6.4–11.2 mg/dl; chicken: 13.2–23.7 mg/dl)
Hypercalcaemia: ovulation (physiological), dehydration, bone tumour
Hypocalcaemia: muscular spasm, seizure, steroid therapy
Hypernatraemia: salt poisoning in wild birds, feeding of excess salt with feed (peanuts, potato crisps) in pet birds
- (b)Different serological, immunological and molecular biology based tests can be performed for detection of avian infections, infestations or intoxications (Table 5.3). Lack of species specific reagents (polyclonal and monoclonal antibodies), standardized and reproducible techniques, considerable variations in results between laboratories are the major constraints in diagnostics of avian medicine. All the laboratory test reports should be correlated with signs and symptoms of the birds by the veterinarian.Table 5.3
Diagnostic approaches for avian infections, infestations and intoxications
Antigen detection ELISA, PCR, isolation
Hemagglutination-inhibition (HI), agar gel immunodiffusion (AGID), virus neutralization, enzyme-linked immunosorbent assay (ELISA), PCR, isolation of virus
Beak and feather disease
Haemagglutination inhibition (HI), blocking ELISA, histopathology, PCR
Avian Bornavirus infection (Proventricular dilatation disease)
ELISA, indirect immunofluorescence assay, isolation of virus, histopathology, reverse transcriptase-PCR, radiography
Direct examination, isolation of bacteria, ELISA
Direct examination, micro immunofluorescence (MIF) test, ELISA, CFT, elementary body agglutination test, isolation, PCR
Direct examination, Detection of Cryptococcal antigen by ELISA, isolation, PCR
Direct examination, Capture enzyme-linked immunoassays, PCR
Direct examination, ELISA (antigen capture), immunofluorescence, PCR
Direct Examination, isolation, ELISA, PCR
Direct examination for acid-fast organisms, histopathology, PCR, isolation
Virus neutralization test, plaque neutralization, hemagglutination-inhibition, agar gel immunodiffusion, enzyme-linked immunosorbent assay, isolation of virus, real-time reverse-transcriptase polymerase chain reaction
Isolation of bacteria, rapid whole blood/serum agglutination test, ELISA, PCR
Direct examination, isolation of bacteria
Direct examination with contrast phase microscopy Contrast phase microscopy, dark phase illumination techniques, serum plate agglutination test, PCR, isolation of Mycoplasma
Direct examination, isolation of bacteria
Escherichia coli infection
Isolation of bacteria, PCR
Wet mount with 10% KOH, histopathology, isolation, PCR
Direct examination by Gram’s staining, histopathology
Visualization of sarcocystis in the muscles after post-mortem, muscle biopsy, indirect immunofluorescent assay, PCR
Examination of faeces for oocysts or macrogametes
Detection of thick shelled Ascarid eggs in faecal sample
Isolation of virus in cell lines, demonstration of virus in clinical samples by electron microscopy, detection of viral DNA by PCR or real-time PCR
Psittacine adenovirus infection
Isolation of virus, electron microscopy, PCR
Psittacine poxvirus infection
Clinical symptoms, electron microscopy, detection of Bollinger body in tissue samples and Psittacine Poxvirus specific-PCR
Avian Polyoma virus infection
Electron microscopy, virus neutralization test, immunofluorescent antibody staining, in situ hybridization, PCR
Psittacine Papillomavirus infection
Presence of typical cauliflower like growth in cloaca or oral mucosa, histological examination of biopsy samples, in situ hybridization, PCR
Avian Reovirus infection
Isolation of virus, electron microscopy, immunofluorescence staining, PCR
Isolation of virus, electron microscopy, pan-coronavirus reverse transcriptase-PCR.
Direct examination, histology, Modified agglutination test, T. gondii-specific PCR
Usutu virus infection
Isolation of virus, immunohistochemical (IHC) staining, PCR
West Nile virus infection
Isolation of virus, immunohistochemical (IHC) staining, PCR
Detection of lead content in blood, radiography of enteric tract
Determination of zinc concentration in blood, histopathological examination of pancreas
History, plasma cholinesterase assay
Polytetrafluroethyelene (Teflon) toxicity
5.6 Diagnostic Techniques in Avian Clinics or Hospitals
Digital radiography is a useful diagnostic technique used currently in avian medicine for rapid detection of underlying hidden etiology. It is easy to perform in birds due to smaller size and one exposure is sufficient to take the image of the whole body. Avian air sac system acts as a negative contrast to the organs which make the interpretation easier for the radiologists. Other than high power X-ray apparatus, film-screen combination and developing system, expert technician is required for interpretation of avian radiographic images. Short exposure times (0.015–0.05 s) are recommended for taking avian images because image quality is detoriated due to high respiration rate of the birds even under anaesthesia. For taking images of internal organs and skeleton of birds, rare earth screens and mammography screens are recommended, respectively. In general, fine film screen combinations are preferred for avian images.
5.6.2 Ultrasonography (USG)
In ultrasonography, the image is produced with transmission and reflection of sound waves. In birds, use of USG is limited because the ultrasound cannot invade a gas filled air sac. Other hindering factors include circulatory and respiratory distress of birds during examination, application of coupling gels to make contact between transducer and avian skin, and lack of experienced veterinarians. Fasting of the birds for 2–4 h is required before USG examination. Confirmation of hepatomegaly, cardiac disease, disorders of kidney and reproductive tract, and ascites is possible through USG examination.
5.6.3 Computed Tomography (CT)
This technique generates a cross-sectional image for accurate visualization through the use of X-rays. General anaesthesia is required and the whole procedure takes 10–15 min time to be completed. Examination of avian skull, sinuses and lower respiratory tract is possible through CT.
5.6.4 Magnetic Resonance Imaging (MRI)
This technique also generates cross sectional images through strong external magnetic force. MRI can detect the presence of caseous plug, granuloma, mucocele, polyp in brain, spinal cord, coelomic organs and upper respiratory tract. Detection of accurate location of these lesions helps in surgery. MRI takes longer time than CT for examination.
In larger birds (1 kg or more), images are taken after injection of non-ionic iodinated contrast medium (0.8–1.2 ml/kg body weight) into subarachnoid space at thoraco-synsacral junction. The technique is indicated for detection of compressive and traumatic lesions in the spinal cord.
Echocardiography provides useful information of cardiac function and structure of the heart. Earlier it was difficult in birds due to presence of air sacs which block the passage of ultrasound waves. Currently echocardiograph machines with advanced technology (7.5 MHz or higher frequency, doppler function, more than 100 frames/s) is used successfully in avian medicine. Fasting for 2–12 h (psittacine, pigeons) or longer period (raptors) is recommended before echocardiography. Food filled enteric tract may create an obstruction between the machine and heart. Ventromedian (psittacines, raptors) and parasternal (pigeons) approaches are followed for avian echocardiography.
5.6.7 Electrocardiography (ECG)
ECG is used to observe cardiac function during anaesthesia and for recording cardiac stages (systole, diastole). Use of ECG in avian medicine is not frequent due to difficulties in making connection of leads with skin of the birds, lack of reference values and alterations of ECG values under stress or anaesthesia.
Avian endoscopy helps the clinicians to examine internal organs (lungs, air sacs, heart, intestinal tract, liver, kidneys, adrenal glands, spleen, pancreas, gonads, oviduct, and shell gland) through a small and single incision. Endoscopy of oral cavity also allows examination of esophagus, crop, proventriculus, glottis and trachea. In addition to physical examination, clinicians can collect tissue biopsies from vital organs, coelomic musculature and abnormal soft tissue structures.