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Part II :
Pathological Specimens
17.7 Introduction:
17.7.1 (a) When any abnormal condition is observed, the nature and significance of which cannot be determined by macroscopic examination of the carcass and/or viscera, samples may be taken for further evaluation.
(b) Evaluation may be performed in the Meat Division laboratory at the premises or at an Animal Health Laboratory.
(c) In certain cases samples should also be sent to Animal Health Laboratories to confirm diagnoses, or as part of a disease control programme.
(d) For requirements for submission of samples to Animal Health Laboratories read these instructions in conjunction with the Animal Health booklet 'Specimens for Laboratory Examination - Selection and Submission'.
17.7.2 An inspector shall inform the company area foreman, or his deputy, before taking samples of any flesh products, byproduct, ingredient, etc. for examination. (This does not apply to meat monitoring.)
17.7.3 All samples must be taken and handled aseptically. Remember these are pathological specimens and YOU, the sampler, may be at risk unless you exercise strict hygienic techniques. The same applies to packing and despatch (see 17.15).
17.7.4 (a) Specimens should normally be saved for:
bacteriological and histological examination.
(b) Replacement plastic specimen jars are provided by the Animal Health Laboratories when returning sample boxes. Obtain additional supplies, if necessary, from the Regional Office, Ministry of Agriculture and Fisheries.
(c) Each individual sample shall be put into a separate plastic jar. Thus the set of samples required from one case will all be in separate jars.
17.8 Specimens for Bacteriological Examination:
17.8.1 Material for bacteriological examination should be taken aseptically, to avoid contaminants masking the findings.
17.8.2 The knife or scissors and forceps used for collecting the samples must be sterilised both before and after use.
17.8.3 Specimens should be 30-40 mm in diameter (hen's egg size) or larger if necessary, e.g. a complete node and its fat cover. Always include the edge of the lesion.
17.8.4 (a) Whenever possible specimens should be complete, i.e. not cut. If an incised specimen has to be sent (e.g. an incised tuberculous node) once the lesion has been exposed, it shall not be further cut or damaged.
(b) A sample is unsatisfactory for microbilogical examination when it has been repeatedly handled or used as a demonstration specimen.
17.8.5 Adequate cooling of samples for bacteriological examination is essential.
(a) They should be chilled and then despatched immediately to the Animal Health Laboratory provided they will not remain in transit over the weekend or be otherwise detained.
(b) Samples awaiting suitable delivery arrangements must be held at a temperature slightly above freezing point, or frozen, until they can be safely despatched (in range +2 to -3 °C). This is because decomposition is rapid at room temperature.
(c) Deteriorated specimens are of little or no value for microbiological examination.
(d) Include sections in 10% formalin for histological examination in case nothing is found in the fresh specimens, as histological findings assist in arriving at a diagnosis.
17.9 Specimen for Histological Examination
17.9.1 Formalin should be used for the preservation of material for histological examination.
17.9.2 Material which has been frozen either before or after placing in formalin is useless for histology as the ice crystals formed damage cell structure.
17.9.3 Samples should be fixed in 10% formalin.
17.9.4 Commercial formalin is 40% formaldehyde gas in water, and must be diluted with water before use. Undiluted it causes great hardening and shrinkage of the tissue, rendering specimens useless for histological examination.
17.9.5 Use 1 volume of 40% formaldehyde (commercial formalin) plus 9 volumes of water as fixing solution. Stock solutions can be neutralised by adding a few marble chips to the container.
17.9.6 Cut the tissue to be examined into slices between 5 and 10 mm thick. Include the edge of any lesion so that both normal and abnormal tissue is seen.
17.9.7 Jars should hold at least 10 times as much 10% formalin solution as tissue.
17.9.8 Brain samples should be either:
(a) fixed whole in a jar; or
(b) cut down the midline - to allow easy removal from the jar.
Don't cut the brain into pieces or slices as the tissue is soft and much of the histological structure will be lost.
17.10 Blood:
17.10.1 In some cases blood smears, serum, or whole blood are required.
17.10.2 Preparation of Blood Smears:
(a) In the preparation of a blood film it is essential that the slide is clean and free of grease.
(b) A convenient 'spreader' can be made from a glass slide by cutting off one corner (see Fig 1). If a normal slide is used the film is spread from edge to edge rendering the smear dangerous to handle and/or cause technical problems with the examination of leukocytes. A properly prepared film should have sufficient clear glass all around it to allow the slide to be safely picked up by the edge (Fig 2).
(c) Place a drop of blood near the-end of the slide (direct from the blood source or from the point of a sterilised knife).
(d) Take the 'spreader' at an angle of 30 ° and draw it backwards along the slide to touch the blood (Fig 3). Allow the blood to run along the bottom edge of the spreader. Move the spreader forwards in a smooth glide along the slide (Fig 4). Allow to dry in the air.
No further treatment of the slide is necessary if it is for studying normal blood constituents e.g. erythrocytes and leucocytes, but the slide must be kept absolutely dry and packed carefully, to avoid scratching and damage to the smear.
(e) Where bacteriology is required e.g. suspected Anthrax cases, fix the smear by passing the slide over a bunsen flame so that the heat of the slide bottom is just bearable to the back of the hand.
(For Anthrax procedure see Manual 11, "Contagious and Exotic Diseases").
17.10.3 Collection of Blood Samples:
NOTE: Blood samples should be collected at the time of sticking.
(a) Clotted Blood, for serum:
(i) Collect in plain 'vacutainers' or equivalent evacuated tubes available from the regional office of the Ministry of Agriculture and Fisheries.
(b) Unclotted Blood:
(i) Use vacutainers containing EDTA (an anti-coagulant), obtainable from the regional office.
(ii) Gently invert to and fro for 30 seconds immediately after collection.
(iii) Leucocytes deteriorate rapidly in anti-coagulant treated blood. If haematological examination is required send a separate blood smear, not fixed.
17.11 Urine
17.11.1 For bacteriological examination - collect 20 ml aseptically.
17.11.2 Leptospires disintegrate rapidly in unpreserved urine. For detecting leptospires, preserve 20 ml urine with 1 ml of 10% formalin.
17.12 Specimens Required:
17.12.1 Suspected Exotic Vesicular Diseases (e.g. Foot and Mouth Disease, Swine Vesicular Disease).
On no account is any sample from a suspect exotic vesicular disease case to be sent to an Animal Health Laboratory by Meat Division staff [refer to Manual 11, Part I Foot and Mouth Disease 11.6(a)].
17.12.2 When septicaemia, pyaemia, or food poisoning organisms are suspected, take the following specimens for bacteriological and histological examination:
(a) Muscle (preferably a piece of extensor or flexor muscle from each of the forequarters i.e. foreshank).
(b) Spleen: the size will depend on the species concerned, e.g. lamb - 1 whole; ox - one end approx. 150 mm in length.
(c) Lymph node (preferably prescapular and precrural and not incised).
(d) Kidney: part of or 1 whole.
(e) Liver: a piece the size of a hen's egg and hepatic lymph nodes.
(f) Any other part of the carcass or organ showing any lesion which a veterinarian or inspector in charge thinks may contain the relevant organism.
17.12.3 For enteritis and suspected salmonellosis take approximately 250 mm of small intestine, securely tied at both ends to trap the intestinal contents, or -
10-20 ml of intestinal contents from the caecum or colon.
Send the corresponding mesenteric nodes in a separate specimen jar. For salmonellosis, rib samples and the gall bladder are also required. (See Manual No.11, "Contagious and Exotic Diseases").
17.12.4 For information on the samples required for specific diseases refer to the Animal Health booklet "Specimens for Laboratory Examination - Selection and Submission".
17.12.5 Mineral Estimations for Deficiency Diseases Diagnosis:
(a) Liver or other samples may be sent to Animal Health Laboratories for copper or other mineral level estimations if so requested by Animal Health, Private or Club Veterinarians; but NOT if requested by the owner of the stock.
17.13 Identification of Samples:
17.13.1 All specimens shall be properly identified and recorded, i.e. the MD reference number, and a description of the contents are to be annotated on every jar - e.g. MW33/75 Fixed lamb liver.
17.14 Report Forms for Pathological Specimens:
17.14.1 Form Ag.L.47 (obtainable from regional offices of the Ministry of Agriculture and Fisheries) is to accompany each set of specimens despatched to Animal Health Laboratories.
17.14.2 All entries must be clearly printed in BLOCK letters, using a BLACK ball-point pen, as the forms are photocopied.
17.14.3 Enter the name and address of the submitter in the top left-hand corner (the submitter is the Supervising Veterinarian of the works, or MI in charge of abattoir).
17.14.4 Enter full name and correct address of the owner of the stock.
17.14.5 Other details are as set out in the explanation on the Ag.L.47 pad.
17.14.6 The case history is to be as full as possible, also describe ante and post-mortem findings fully.
17.15 Despatch of Samples to Animal Health Laboratories:
17.15.1 Use sample boxes with the lidded tin inside which are provided by the Animal Health Laboratories:
(a) The Ag.L.47 is to be put on top of the tin lid, not inside with the sample.
(b) A separate specimen box should preferably be used for each case.
(c) If (b) is impracticable, all specimen jars from one case should be put into a plastic bag which is then tied and labelled with the case number.
(d) Put a frozen coolant pad into each tin when packing the specimens for despatch.
17.15.2 Specimens must be packed so that they do not contaminate each other, or leak in transit. On no account are pathological specimens to be packed direct into a plastic bag only.
17.15.3 Meat Division staff must take great care to ensure that the outside of pots and jars and the Ag.L.47s are clean and not contaminated.
17.15.4 Obviously submission of specimens in plastic bags, paper towels, or similar packaging is a health hazard to ALL who may handle them e.g. railway employees, laboratory technicians, etc.
17.16 Method of Despatch:
17.16.1 (a) Normally express rail or local transport arrangements.
(b) Air transport is expensive and generally inconvenient for the laboratories. Where air services are used inform the laboratory by telephone of the flight number so the specimens can be collected from the airport without delay.
(c) Postal despatch of goods rail must NOT be used.
(d) If in doubt, contact your local Animal Health Laboratory as to the most suitable system.
New Zealand Food Safety Authority
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NEW ZEALAND
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