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Avian Hematology
Diagnostic Pathology of Zoo animals Gerry M. Dorrestein NOIVBD Veldhoven (NL) VFU-FVL Brno (CZ) Internacionalizace výuky veterinární medicíny jako cesta na evropský trh práce projekt è. CZ.1.07/2.2.00/28.0288 Diagnostic Pathology of Zoo animals Gerry M. Dorrestein NOIVBD Veldhoven (NL) VFU-FVL Brno (CZ) ZooAnimal Pathology 2 Introduction Zoo animals • 4 000 • 9 000 • 4 000 • 20 000 • ????? mammalians different birds reptiles and amphibians fish species invertebrates ZooAnimal Pathology 3/69 What is Diagnostic (Clinical) Pathology? • Use of dead animals and abnormal tissues/fluids to find out what disease problem is going on. • Microscopic examination (cytology, histopathology) of swellings, abnormal tissues, “fluids” from live animals 05/10/13 When tissue and fluid examination in live patients? • Swellings and tumours – find out what tissue – find out if benign or malign – complete surgically removed? • Abnormal tissues e.g. skin biopsies – find the cause of the lesion • fluids from cysts, abscesses, blood, etc – Find info about aetiology or organ function • Abnormal organ-tissue at surgery 05/10/13 Sample collection in live patients • But what you collect is what you get!! • Think-ahead what answer to expect from the sample • Use a sample to CONFIRM your thinking ZooAnimal Pathology 6/69 Fine Needle Biopsy • Fine needle ♦ ≤ 22 gauge ♦ +/- syringe ♦ Redirection ♦ Forward cutting motions What’s the Target? Absence of proof is not proof of absence. Introduction PME • Not all patients will survive! • Therefore we do a post mortem exam • The key of a PME for a clinician is to find an explanation for the clinical findings • And vice versa: the clinical finding indicates pathological changes ZooAnimal Pathology 9/69 Why a post-mortem examination? (PME) • • • • • • • • • To find out the cause of death To diagnose the problem in a collection As sentinel animals in a group To confirm the clinical diagnosis To evaluate/understand therapeutic failure To prevent spreading of an infectious disease New emerging diseases For anatomical-clinical study or just curiosity For recognition under the Balai legislation (directive 92/65/EEC transport between zoos) ZooAnimal Pathology 10/44 Why post-mortem examination? (PME) • The main issue is to understand what is going on!!! • Understand what you see…. • Know what to expect based on clinical information…… 05/10/13 Why do animals die? Four basic essential needs Why do we live, technically?? What are the basic requirements for cell function?? ZooAnimal Pathology 12/69 Basic requirements for life 1. Oxygen 2. Energy 3. Water Results in membrane functionstability 4. Toxins attacking membranes ZooAnimal Pathology 13/69 Basic requirements for life Therefore Evaluate your findings related to these basic requirements ZooAnimal Pathology 14/69 What do we have before we start the PME ? • • • • • History Signs and symptoms Imaging information Laboratory results Treatment results >>>> = INFORMATION ZooAnimal Pathology 15/69 You also have knowledge = your veterinary education, etc This should trigger your medical and pathophysiological thinking!!! Resulting in: • Information hypothesis • Knowledge • Imagination However too many clinicians get stuck with axioms ZooAnimal Pathology 16/69 The solution Make yourself understand the problem in the animal Use educated imagination/phantasm ZooAnimal Pathology 17/69 How are you supposed to do it?? • • • • • • Analyze the history (use books/Internet) 50% of your diagnosis is in the anamnesis!! Find and explain (understand) changes at PME Formulate a theory!! (hypothesis not diagnosis) “I-do-not-know” is not acceptable Find the truth behind the hypothesis (could be a lie) • Use direct tests (in the necropsy room) • Use bacteriology, histology, etc to confirm • Formulate keywords (for google, pubmed, etc) ZooAnimal Pathology 18/44 Example Siberian Chipmunk (Burunduk) (Tamias sibericus) Hypothesis ? ZooAnimal Pathology 19/44 Use direct techniques Cytology smear Wet mount ZooAnimal Pathology 20/44 Auxillary techniques Confirmation with histology Diagnose: Capillaria hepatica ZooAnimal Pathology 21/44 Preparation before starting the PME • • • • Collects as much history as possible Evaluate ALL clinical data Study again X-rays and/or other imaging Formulate the diagnosis or a DDx (hypothesis) • Realise what samples or material you need for confirming the different possibilities • Organise TIME ZooAnimal Pathology 22/44 Preparing for a PME • Study the history and think in DDx • BUT avoid preconceptions that may limit information and specimen collection • Take photographs • Prepare to collect complete set of tissue- and blood samples from the carcass ZooAnimal Pathology 23/44 Get experience in PM, cytology and histology of exotic animals ZooAnimal Pathology 24/69 Basic lay-out for a necropsy of a small animal 1. Sharp scalpels 2. Forceps and scissors 3. Bone cutters 4. Formalin 5. Alcohol 6. Saline 7. Containers 8. Glass slides 9. Burner ZooAnimal Pathology 25/69 Basic lay-out for a necropsy of a small animal 10.Culture media 11.Oses 12.Cleaning fluid 13.Small plates 14.Marker 15.Stickers 16.Ruler 17.Checklist 18.Paper towels 19.The animal ZooAnimal Pathology 26/69 PME small animals ZooAnimal Pathology 27/69 Autopsy site large animals ZooAnimal Pathology 28/69 Notifiable diseases • When a legally notifiable disease is suspected or encountered during the autopsy contact the appropriate national veterinary authorities (directive 92/65/EEC for Zoos) • These include: FM disease, malignant catarrhal fever, tuberculosis, salmonellosis, yersiniosis, influenza, psittacosis, Ncd, etc • See OIE Lists A and B (http:/www.oie.int) • Handbook of Infectious Diseases of the EAZWV ZooAnimal Pathology 29/69 Personal safety precautions • Consider zoonotic or notifiable disease • Some zoonoses: – – – – – – – ??? Anthrax (ungulates and carnivores) Rabies (esp. bat lyssavirus) Echinococcus sp. (carnivores) Tuberculosis (all species, esp. mammalian) Psittacosis (ornithosis) Tularaemia, hantavirus (rodents) Hepatitis B and other viruses (monkeys) ZooAnimal Pathology 30/69 (Personal) safety precautions Wear protective clothing Samples should be collected, stored and transported safely to prevent escape of infective material ZooAnimal Pathology 31/69 Protective clothing • • • • • Rubber boots Rubber or plastic gloves Rubber or plastic apron Overalls or special clothing Face mask (incl goggles to cover eyes) ZooAnimal Pathology 32/69 Post-mortem procedures There is no standard protocol. Be consistent Make a checklist Be systematic Start ASAP ZooAnimal Pathology 33/69 Basic Post-Mortem procedures Handbook or CD of Infectious Diseases of the EAZWV • • • • • History External examination Internal examination Collection of samples (per organ system!) Laboratory activities – (faeces, blood, cytology) • Preservation and distribution of samples • Finalising the report by making a hypothesis or preliminary diagnosis ZooAnimal Pathology 34/69 External examination • Ruminants check for bloody discharges • FIRST make two blood smears to check ANTRAX • Stain with quick-stain • In horses, pigs and carnivores take cut surface of a superficial lymph node • Wait with the necropsy for the results ZooAnimal Pathology 35/69 Sample collection keep it simple!! ZooAnimal Pathology 36/69 Routine Laboratory Testing Faeces (crop,stomach,intestine), urine, se- & excreta Macroscopy Microscopy Colour, consistency, aspect, smell Wet mount (NaCl 0.9%, amph. 0.6%) Enrichment (coccidia, worm-eggs) Stained smears, cytology (Abnormal) tissue Cytology Histology Electron microscopy (EM) Microbiology, serology, molecular biology (PCR) ZooAnimal Pathology 37/69 Routine Laboratory Testing CAN PROVIDE QUICK AND USEFUL INFORMATION BUT BE SURE YOU ARE TAKING THE RIGHT SAMPLE/MATERIAL AND BACK-UP THE RESULTS ZooAnimal Pathology 38/69 Simple on-spot techniques • Wet mounts (use saline) • Stained impression smears (=cytology) • Bacteriology / mycology ZooAnimal Pathology 39/69 Wet mounts ZooAnimal Pathology 40/69 Cytology as Instant Diagnostic Tool • • • • • • • Rapid, inexpensive method Easily done in any practical setting But it needs some practicing Microscopic information about a “process” Often an etiologic agent can be found Some diagnosis cannot be made without Does not replace histology ZooAnimal Pathology 41/69 Preparation of the slides ZooAnimal Pathology 42/69 Staining of the impression/smear • • • • • Quick stain (general, invl protozoa) Macchiovello/Stamp (Chlamydophila) Ziehl Neelson (Mycobacteria) Gram stain (differentiation bacteria) Etc. on specific request ZooAnimal Pathology 43/69 Staining of the cytology slide Hemacolor® • • • • • • Dry at the air sol 1: 10-60 sec fixation sol 2: 10 sec till orange sol 3: 10-30 sec till purple-blue sol 4: 4-5 sec buffer (pH 7) no rinsing with water in between ZooAnimal Pathology 44/69 Staining of the slide Stamp + stamp- or Macchiavello’s stain • Specific for Chlamydophila sp. • sol 1: 5 min, rinse with water • sol 2: 5-10 sec de-staining, rinse with water • sol 3: 8 sec counterstaining, rinse with water ZooAnimal Pathology 45/69 Example 1 kakariki sinusitis • Kakariki with a sinusitis • Weeks of Ab treatment no success • Sample of sinus fluid • Stained with Hc • And culture ZooAnimal Pathology 46/69 Example 2 Black tufted ear marmoset Clinical history • Constipation • Clinically suddenly weak • Mass in abdomen • Explorative surgery • Euthanasia Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 47/69 Example 2 Black tufted ear marmoset PM exam • Tumour-like lesion with severe adhesions with abdominal wall • Involving intestines • Multi micro-lesions in liver Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 48/69 Example 2 Black tufted ear marmoset Laboratory results • Fecal exam: negative • Cytology: incl lungs • Bacteriology liver and lung – E. coli – Aeromonas hydrophila Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 49/69 Example 2 Black tufted ear marmoset Main macroscopic findings: • Intestinal tumour • Multifocal hepatitis Preliminary Diagnosis • Intestinal obstruction due to tumour-like structure and bacterial sepsis Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 50/69 Example 2 Black tufted ear marmoset Additional info with histology • Infiltrating carcinoma • Liver necrosis with many bacteria • Acute pneumonia after food aspiration Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 51/69 Example 2 Black tufted ear marmoset Additional info with histology • Infiltrating carcinoma • Liver necrosis with many bacteria • Acute pneumonia after food aspiration Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 52/69 Example 2 Black tufted ear marmoset Additional info with histology • Infiltrating carcinoma • Liver necrosis with many bacteria • Acute pneumonia after food aspiration Courtesy to Dr Sonja Luz, Singapore Zoo ZooAnimal Pathology 53/69 Basic microbiological isolation but first cytology!! Aerobic culture •Bloodagar •Selective agar (Gram -) •Serumbroth •Enrichment fluid (salmonella) •Media for fungi •Incubator 37C ZooAnimal Pathology 54/69 Necropsy Lung bacterial pneumonia Anaerobes Not grown!! ZooAnimal Pathology 55/69 Another example Culturing result ZooAnimal Pathology 56/69 Result Bacteriology Laboratory E. coli Sensitive to Not sensitive to • • • • • • • • • • • • • Amoxy Clav Cefquinome, Cephalexine Cefoperasone Colistine, +/- Doxy Florfenicol, Furazolidone Lincomycine, Neomycine Tulathromycine, Ceftiofur Ampi, Amoxy Enroflox, Flumequine Tetrac TMPS Tylosine Difloxacin What is your choice and why? ZooAnimal Pathology 57/69 Additional sampling • Fixation of tissues in formalin 4% • Histology • Tissue suspension in alc 70% • PCR • Liver, spleen, kidney, etc -20% • Virus, tox • • • • Specific sampling related to DDX Sampling related to research Sampling related to stud-book Sampling for eductional projects ZooAnimal Pathology 58/69 External examination • Condition of the skin, fur or feathers • Record broken limps or abnormalities • Examine for external parasites – Around muzzle, eyes, ears and genitals – On the neck, brisket, tail switch, axillae, groins and hoof clefts • Examine mouth/beak for condition oral mucosa, tongue and teeth ZooAnimal Pathology 59/44 External examination sika deer BCK = MCF ZooAnimal Pathology 60/44 External examination Macaca mulatta seborrhoea ZooAnimal Pathology 61/44 External examination Macac mulatta herpesvirus ZooAnimal Pathology 62/44 External examination Macaca sp. myxoma ZooAnimal Pathology 63/44 External examination Macaca sp. cowpoxvirus ZooAnimal Pathology 64/44 External examination Gorilla candidiasis ZooAnimal Pathology 65/44 External examination Rabbit myiasis ZooAnimal Pathology 66/44 External examination Oryx gazella chronic wasting ZooAnimal Pathology 67/44 External examination Mara dental bridging ZooAnimal Pathology 68/44 External examination Rabbit ear mites ZooAnimal Pathology 69/44 Eclectus parrot PBFD = circovirus ZooAnimal Pathology 70/44 External examination Cormorant plastic six-pack ZooAnimal Pathology 71/44 External examination avipoxvirus ZooAnimal Pathology 72/44 External examination bumble foot ZooAnimal Pathology 73/44 External examination Metabolic Bone Disease ZooAnimal Pathology 74/44 External examination mycotic inflammation ZooAnimal Pathology 75/44 External examination stomatitis mouth rot ZooAnimal Pathology 76/44 External examination chronic dermatitis ZooAnimal Pathology 77/44 External examination shark skin irritation ZooAnimal Pathology 78/44 Example from Brno CZ ZooAnimal Pathology 79/69 Gepard Brno 07-04 Collecting cytology sample ZooAnimal Pathology 80/69 Gepard Brno 07-04 Staining Hemacolor® ZooAnimal Pathology 81/69 Gepard Brno 07-04 Cytology (HC) ZooAnimal Pathology 82/69 Gepard Brno 07-04 This is what we are looking for!! Ideal would be a biopsy to confirm the preliminary diagnosis Eosinophilic granuloma ZooAnimal Pathology 83/69 Success and enjoy the PME! ZooAnimal Pathology 84/69 Questions? ZooAnimal Pathology 85/69 Thank you ZooAnimal Pathology 86/69
