Post mortem examinations and cytology in avian veterinary practice

Post mortem examinations and cytology in avian veterinary practice Edmund Flach Veterinary Department, Zoological Society of London, Regents Park, London NW1 4RY POST MORTEM EXAMINATION OF BIRDS Source of carcasses for post mortem examination (PME) Exotic pet birds; owner with one to a few birds / serious keeper +/- breeder / importer. Birds of prey; hobby falconer / large-scale falconer & B.O.P. centre Ratites; farms / owners keeping rheas Domestic birds, poultry and waterfowl; owner with one to a few / backyard hobby / commercial enterprise. Wild birds: found dead and handed in / found injured and euthanased / mortality outbreak Zoos: small, mixed collections / large avian collections / medium to large zoos Reasons for PME a) Identification of cause of death, or cause of clinical disease responsible for the decision to euthanase b) Confirm the presence or absence of pathology & explain lack of response to treatment (if given). c) Identify aetiological agents and predisposing factors for diseases. d) Check for any un-recognised diseases or pathogens (early warning for others) e) Opportunity to investigate comparative anatomy, e.g. to improve radiographic or surgical technique Staff health and safety DO NOT PUT YOURSELF, OR YOUR STAFF, AT RISK IF IN DOUBT, SEND CARCASS TO A SPECIALISED LABORATORY WITH MICROBIOLOGICAL SAFETY CABINET ETC. Major zoonoses include: Chlamydiosis Avian influenza, especially H5 N1 Mycobacteriosis Salmonellosis Campylobacteriosis

Set protocols for dealing with major bird groups and ante-mortem clinical signs, eg. a) All psittacine birds to be sent away for PME (check with external labs to find which will take these) b) All wild waterfowl to be sent to the local VLA (check that they are continuing to do wild bird PMEs for AI monitoring) c) Any bird showing unexplained respiratory signs to be sent away for PME. d) Any birds weighing more than a threshold (dependent on the available space, facilities and equipment at the practice) to be sent away for PME Set protocols for safe handling of carcasses, examination and sample-taking, and disposal (see below). Only trained personel to perform PMEs (vets +/- vet nurses given appropriate training in dissection and sample collection). Clear designation of space for PMEs and differentiation between clean and dirty areas, equipment etc. Examination area and equipment Ideally a dedicated post mortem room Otherwise use an area within a laboratory or similar room which does not need to be used when a PME is being undertaken, and which can be cleaned and disinfected. Good idea to do PMEs within large, deep trays; easy to differentiate between clean (outside tray) and dirty (inside). Chopping board and/or cork board and pins. Use good quality dissection instruments: scissors (pointed and blunt, small, medium and large, bowel forceps small and large), forceps (blunt and rat-toothed, small, medium and large), scalpel handles and disposable blades (standard, pointed and heavy-duty), bone cutters, bone saw (for larger specimens). Weighing scale(s) to cover the range of birds likely to be seen, plus eggs, organs etc. Best to have several: pan scales (e.g.10mg to 100g for eggs, embryos and small birds, 1g to 1kg for medium-sized birds, 100g to 25kg) +/- spring balance(s) for medium and large birds (eg.1 to 100kg). Ruler, measuring callipers (to 0.1mm), tape measure. String. Camera capable of close-focus and macro. PM record sheets, pens, pencils, indelible marker pens Microbiology swabs (plain and with transport medium) Sample containers (sterile 7m bijous, sterile 30ml universals, larger pots for histopathology). Blood/fluid tubes (plain, EDTA and heparin) Glass microscope slides with frosted ends, plus coverslips (small and large) and slide holders. 10% buffered formalin for histopathology. 70% ethanol (for parasites)

Normal physiological saline, or phosphate-buffered saline, for wet preparations. Plastic bags, many different sizes, including some with sealable openings Post mortem examination Follow a set format based on a PM form (see example; Appendix 1). Always keep meticulous records, but if the case has any legal ramifications get further information and advice regarding forensic pathology. History Check that you have details of the bird (species, age, sex, identification etc.) and owner. Check for a transponder microchip if expected (or if any suspicion about the bird s identity) If a wild bird, confirm the place where it was found. Check any clinical history and diagnostic testing done. If any evidence of metabolic bone disease, or history of lead poisoning, or being shot, consider radiography before the carcass is unwrapped. Further radiographs of the skeleton may be done after the PME. External examination Check and record any identification tags, or other identification markings. Weigh the bird and take appropriate measurement (check if any are required, such as wing length, beak length etc.). Assess body condition (prominence of the keel, skeletal muscle development, subcutaneous fat stores). Assess state of hydration; but often best done while the skin is dissected free of the underlying tissues (see below). Examine the whole carcass: skin, feathers, beak (open and examine inside), cere and nostrils, eyes, ears, wings, legs and feet, preen gland, tail and cloaca. Check for an external yolk sac in embryos and newly-hatched chicks. Palpate the whole body and manipulate all joints. Take diagnostic samples of any suspicious lesions. Soak the feathers in dilute disinfectant containing detergent to reduce the risk of inhaling feather dust, and feathers getting into the coelomic cavity. The feathers from the ventral body can be plucked to further reduce the problem of stray feathers getting into the coelom. Internal examination Midline skin incision from the base of the lower beak caudally to just cranial to the cloaca. Blunt dissect the skin from the underlying tissues to expose the neck, pectoral muscles and caudal coelomic wall.

Examine the exposed trachea, oesophagus and crop (if present), clavicular air sac (if present) and pectoral muscles. Remove the pectoral muscles from the sternum and ribs. Assess the muscle depth and make several transverse cuts to look for lesions (haemorrhage, necrosis, inflammation etc.). Retain any transponder microchip found and recheck the reading later. If fresh blood is available at this, or any later stage, use a syringe to draw it some up for a fresh blood smear and to save some in EDTA +/- heparinised blood tubes. Cut through the caudal coelomic wall; along the ventral midline from the keel to the cloaca, and laterally just caudal to the last ribs. Examine the viscera and air sacs whilst doing this. If any excess fluid is present, collect some with a syringe and place in plain +/- EDTA tubes. Cut through the ribs ventral to the lungs, using scissors or bone cutters as appropriate. Cut through the ventral attachments of the air sacs and pericardium to the sternum. Whilst holding the body firmly, lift the sternum and ventral parts of ribs and cut through any remaining attachments. In a small bird bend the sternum right back to fracture or dislocate at the tri-osseous joint, whereas in larger birds it may be necessary to cut through the articulations. Assess the positions and relative sizes of the air sacs, heart, liver, proventriculus, ventriculus (gizzard) and coelomic fat bodies. With chicks and embryos, also check the internalised yolk sac. Record any lesions visible. Use blunt and/or sharp dissection to isolate the heart and major blood vessels. Identify the thyroids and parathyroids alongside the carotid arteries at the entrance to the pectoral girdle. Look for evidence of thymic tissue. This is often indistinct in birds, but if fatty deposits in the sub-cutaneous connective of the base of the neck on the left side are taken for histopathology, thymic tissue is often present. Cut through the major blood vessels (retaining the thyroids/parathyroids) and remove the heart. If a clean dissection site is required, vessels should be ligated before sectioning. Isolate the oesophagus from surrounding tissues and cut through it (+/- ligating it first) and draw it caudally whilst detaching it from other tissues. Depending on the importance of examining it in its entirety, it may be cut just caudal to the pharynx and fed through the pectoral girdle before continuing, or it may be cut at the pectoral girdle and the two parts examined separately. Continuing to draw the oesophagus caudally, along with the rest of the gastrointestinal tract, plus liver and spleen, identify all attachments, and then cut through them. Eventually there will just be the rectum extending into the cloaca. Examine the dorsal aspect of the rectum and cloaca for the cloacal bursa (Bursa of Fabriscus) and remove with the rectum +/- cloaca (ligating the terminal rectum if necessary). Separate the liver and spleen, and unravel the gastrointestinal tract. Take particular care to preserve the yolk sac in embryos and chicks. Cut through the submandibular tissues on the medial aspect of the lower beak and mandible to free the tongue and larynx. Free the larynx from the pharynx and oesophagus and separate the trachea along the neck.

Examine the ventral surface of the lungs, then remove by cutting along the medial and lateral aspects, then using a scalpel handle (or similar long, flat implement) to separate them from the thoracic spine and ribs respectively. Identify, and then remove by dissection, the gonad(s) and adrenals from the ventral aspect of the cranial kidney lobes, plus the oviduct if a female. Remove the kidneys by blunt dissection using a scalpel handle (or equivalent). Once the viscera have been removed, standard bacteriological swabs (or tissue samples) are taken from the heart (heart blood inside one of the ventricles) and liver, plus any organs which look congested or inflamed. Use a fresh scalpel blade to make a clean incision, then rotate the blade by 90 degrees to open the tissue for swabbing the exposed parenchyma. Work through the body systems systematically; examine and palpate each part, describe and photograph any lesions, and take appropriate diagnostic samples for microbiology, cytology and histopathology. Standard impression smears of liver, spleen, lung and kidney are made (see below). A sample of faeces, or large intestinal content, is collected for parasitological and bacteriological examinations. Don t forget that avian hearts have a muscular right atrioventricular valve. If unsure about the anatomy of an unusual species, make sure to check before assuming that pathological lesions are present. Examination of the skull may require opening into the nasal cavity (cutting across the base of the beak at the level of the choana is the easiest option), sinuses, and the cranium. To examine the brain, remove the skin from the cranium and then any muscles attaching to the lateral and caudal aspects. The skull can either be separated at the occipito-cervical junction, or dealt with still attached. Cut through the cranium with six cuts in a hexagonal pattern to remove the entire roof (a scalpel and scissors are sufficient for small birds, a hand or oscillating saw may be required for larger ones). Cut through the dura with scissors and reflect it to expose the brain. Swab the dura or meninges if congested; before attempting to remove the brain. Tip the head vertically and ease the brain out with a blunt instrument, cutting the optic and cranial nerves as they appear. Finally, cut though the cervical spinal cord (unless this was done earlier) and place the brain in 10% buffered formalin (at least ten times, and preferably twenty times the volume of the brain). Any musculoskeletal lesions should be examined and sampled. A long bone may be bent and broken to test for strength. Bone marrow may be collected from non-pneumotised bones (eg.tibiotarsus). Joints may be opened to examine articular surfaces and synovial fluid. Examination of eggs. The history should include details of parentage, date laid, number in clutch, incubation details (parental or artificial, if the latter then make and model of

incubator, temperature and humidity data, turning frequency, any unexpected occurrences, such as a power failure), candling details, the date (and stage of incubation) of removal, and reason for removal (e.g. cracked egg, past expected hatching date etc.). The egg should be weighed and measured with callipers (length and maximum width/diameter). The shell should be examined for signs of damage, build-up of calcium, state of cleanliness etc. The shell should be opened at the blunt end with a circumferential incision using scissors. It is advisable to make an initial hole, by drilling into one spot with one arm of a pair of scissors, whilst the egg is in a plastic bag, in case it explodes! Once this is done the egg can be removed and the cut continued. Check whether an obvious embryo is present, and whether it is close to hatching, or even pipping internally (though the air space membrane) or externally (through the shell). Examine the air space and membrane. Cut through the membrane and take a bacteriological swab from inside the egg. Then: a) If there is no obvious embryo present, tip out the contents and search for an embyronic disc or early embryo, plus evidence of vascularisation. If an embryo is present then it should be weighed and measured (crown to rump) and saved in formalin for histopathology (+/- a bacteriological swab from its coelomic cavity). If not, the egg can be recorded as suspected infertile (no embryo found). b) If there is an obvious embryo, but not close to hatch, the contents can again be tipped out and the embryo weighed and measured, but additionally examined more closely externally and internally. A bacteriological swab should be taken from the coelomic cavity and the embryo saved in formal saline. c) If there is a fully-developped embryo present, cut away more of the egg shell so that its position can be determined. The normal hatching position is with the head under the right wing, and the beak points towards the lower slope of the air space. Malpositions include: head over right wing, head under left wing, rotation away from the air space, leg over head and embryo upside-down. The severity of these positions, and likelihood that they were lethal, increases in the same order. Weigh the embryo and measure crown to rump length. Examine the yolk sac and embryonic membranes. Open the embryo and take a bacteriological swab from the coelomic cavity, then examine, +/- sample individual organs. Save representative tissues in formal saline pending histopathology. Examine the inside of the egg shell for any abnormalities. Measure the thickness of the shell at different points if worried about, for example, fragility or excess calcification. Diagnostic sampling Diagnostic tests during the PME: A microscope should be available for examination of samples taken during the PME: feathers, (dry, under a coverslip), skin scrapings (dry or in normal saline for ectoparasites), discharges (for bacteria, fungal hyphae, leukocytes etc.), intestinal contents (food material, fat globules, bacteria, parasites, blood cells)

Diagnostic tests in the practice lab: Impression smears, scraping smears and smears of fluids (blood, discharges, pus etc.) should be air dried, fixed in methanol, and then stained in a Rowanowsky stain (RapiDiff and Diff-Quick are both fine) and/or Gram s, ZN etc. Skin scrapings may be cleared in potassium hydroxide before microscopical examination. Faeces should be examined for parasites as a direct wet preparation (if not already done) and after a concentration technique (saturated salt, Ovassay etc.). Coccidial oocysts can be sporulated in potassium dichromate to check whether Eimerian or Isosporan type. Bacteriological samples may be dealt with if the practice does routine culture and identifications, or sent off to a diagnostic lab. Blood collected into EDTA or heparin can be used for haematological and biochemical testing if sufficiently fresh. Any body fluids should be tested for specific gravity and for ph, protein concentration etc. with a urinalysis dipstick. It can then be centrifuged and a smear made from the pellet to examine for cells and pathogens (RapiDiff or Diff-Quick). Tests to send off: Bacteriology and blood testing, if not available in-house, or if requiring more specialised tests. Virological testing. Histopathology Electron microscopy Toxicological testing Archiving In zoo work, archiving carcasses and tissue samples frozen, plus tissues in formal saline, is an important part of providing a pathological and research resource for the future. With owned animals, it would be necessary to obtain permission before archiving any tissues. Archives are very useful if a new disease is recognised: was it present, but unrecognised in previous cases? Also, to provide control tissues (from birds with known history and PM findings) for comparison with suspected diseased ones. Labelling is vital. Clear, unambiguous, indelible, and preferably in duplicate. Recording of what has been archived is just as important, and should allow rapid recovery of the sample of interest. Waste disposal All sharps should be disposed of in dedicated sharps containers at the end of the procedure. Any unwanted tissues should be double-bagged, along with paper tissues used to soak-up any residual fluids. These are disposed of as clinical waste. Whole carcasses, particularly larger ones, should also be double-bagged, but will need to be disposed of separately, as animal carcasses. All trays and utensils should be washed, and then disinfected.

Use traps in sinks to catch any particulate debris (especially feathers) so that these can be disposed of as clinical waste. Record-keeping and data handling The PM record should be completed and checked at the end of the procedure. All diagnostic samples should be recorded, and their results added to the report. Spreadsheets are useful for logging summarised details of PMs (main details, main findings, samples taken etc.) Word documents are fine for writing reports; a basic format can be set up and used each time. A database is the preferred option for keeping track of large numbers of PMEs. Data can be presented in report style (to avoid the need to keep a separate Word document) and also exported to spreadsheets for analysis. USE OF TISSUE IMPRESSION SMEARS FOR CYTOLOGICAL EXAMINATION Advantages: Easy to do. Ready to read soon after the PME Excellent for examining the cells present in the tissues concerned; cell types and morphology. Identify pathogens present, particularly bacteria and protozoa Identify other material present, e.g. fat, iron (best with alternative stains) Disadvantages You don t see the tissue architecture, CF histopathology Beware artefacts, e.g. stain deposit. Technique Clean two (or more) microscope slides. Cut a small (up to 1cm square) piece of the tissue of interest. Dab one of the cut surfaces repeatedly on tissue paper (a type which does not fray) until blood ceases to be left on the paper. Dab gently 2-3 times on each of the slides. Repeat with other tissues; applying impressions from up to 4 tissues on each slide. Label the slides with the species, PM number, list of tissues, stain required and date. Ensure the slides are air dried. Fix in methanol (or RapiDiff / Diff-Quick fixative). Stain one of the slides in RapiDiff, Diff-Quick, giemsa or equivalent Rowanowskytype stain. Rinse well, dry the back of the slide, tap the slide on its side against tissue paper to remove excess water, then leave to dry.

The other, fixed, slide should also be allowed to dry, and then is kept pending the need for further staining based on the Rowanowsky-stained smear (if bacteria are present then consider a Gram s stain, if un-stained bacilli then ZN, etc.) Examine the smears under low power to find suitable areas to examine (not too dense and a good number of cells present), then add immersion oil and examine at x100 with an oil immersion lens. Take care not to use a dry lens with oil, but if you do by mistake clean the oil off immediately with lens tissue, followed by a swab dipped in xylene, chloroform or acetone. Cytological examination Identify normal tissue cells (e.g. hepatocytes in the liver) and assess their morphology (e.g. variation in size, presence of vacuoles). Identify, and quantify, any abnormal cells (e.g. leukocytes), and also any pathogens. Report on all findings, and added a comment putting the findings in context with the gross PME findings, and any other diagnostic test results. The more smears you examine, the better you will get at cytology!! Cleaning after examination Impression smears can be cleaned by a) placing a strip of tissue paper on the slide to absorb the excess oil, b) adding a drop of chloroform, xylene or acetone to one end of the tissue paper strip and then c) pulling the other end and drawing the solvent over the part of the smear which had the oil on. THESE SOLVENTS ARE HIGHLY INFLAMMABLE AND TOXIC. THEY SHOULD ONLY BE USED BY TRAINED STAFF WORKING IN A WELL- VENTILATED AREA WITH NO NAKED FLAMES ANYWHERE NEAR. PLEASE CHECK COSHH GUIDELINES AND CARRY OUT A RISK ASSESSMENT BEFORE USING THEM. FURTHER READING Latimer, K.S. & Rakich, P.M. (2007). Avian cytology. Veterinary Clinics. Exotic Animal Practice, 10: 131-154. Ritchie, B.W., Harrison, G.J. & Harrison, L.R. (1994). Necropsy examination. Chapter 14. In: Avian Medicine: Principles and Applications. Wingers Publishing Inc., Lake Worth, Florida. Pp.355-379. Appendix 1. Post mortem examination form used at the Zoological Society of London. This form is used for all taxa, therefore some questions and tick-boxes do not apply to birds.

POST MORTEM FINDINGS WildPath No: interim final PME No.: / /09 Common Name: Clinical No.: / Scientific Name: Weight: g kg Section: Sex: M F U ARKS No. Other ID Born / Laid: / / Arrived at collection: / / Age: Died / Found dead / Euth Date: / / Time: am / pm PME Date: / / Time: am / pm Photo: dig./ana. X-RayNo.: CARCASE STORAGE: none refrigerated frozen other CARCASE CONDITION: fresh autolysed mummified other PHYSICAL CONDITION: normal fat thin emaciated other HYDRATION STATUS: normal dehydrated undetermined other 1. NAD NE skin /apps 2. NAD NE sensory 3. NAD NE muscular 4. NAD NE skeletal 5. NAD NE cavities 6. NAD NE alimentary 7. NAD NE liver 8. NAD NE respiratory 9. NAD NE cardiovascular 10. NAD NE lympho-retic. 11. NAD NE urinary 12. NAD NE endocrine 13. NAD NE reprod. 14. NAD NE nervous 15. NAD NE spinal cord Gross Diagnosis: Pathologist Prosector

Outside Specimen Requests: DNA Carcase saved frozen: Other (bone/shell etc) Parasitology (if more than one sample, then assign number to source, test and result) Source: faeces stomach duodenum jejunum ileum caecum colon skin hair other Test: flotation direct wet mount MZN stain Giemsa Stain other Result(s): positive negative uncertain other Bacteriology/Mycology (if more than one sample, then assign number to source, test and result) Sample type: swab tissue content exudate touch other Site: Test: Gram Stain ZN Stain MZN Stain Culture Sensitivity Result(s): Cytology Fluid: Tissue: Test: wet mount RapiDiff Giemsa other Result(s): Urinalysis from bladder from floor/table Result: Glucose Bilirubin Ketones SG Blood ph Protein Urobilinogen Nitrate Leucocytes Microscopy: done not done Samples stored in formalin/70%ethanol: SAVE ONLY TRIM / / DUPS Special stains etc Skin Gizzard Gall Bladder Thymus Adrenal Nerve Eye Duodenum Pancreas Spleen Pituitary Muscle Jejunum Trachea Cloacal Bursa Ovary/Ovaries Bone Ileum Lung Kidney Uterus Oesophagus Int NOS Gill Head Kidney Testes Stom/Abo/PV Caecum Air Sac Urinary Bladder Prostate Rum/Ret/Om Colon Heart Thyroid Brain Stom NOS Liver Aorta Parathyroid Spinal Cord Other Samples submitted for testing / / saved Frozen Samples: B / U liver kidney lung spleen stom. Cont brain other Fixed Samples: parasite other fixative Test: virology toxicology parasitology iron other Laboratory: Result(s)