SE 1929: Studies of Experimental BSE in Suceptible Sheep

SE1736 CSG7APPENDIX 1

SE 1736: Necropsy Protocol I

Check eartag to ensure correct animal has been selected before starting any procedure

From female animals please sample the following tissues

Udder, Teat, Endometrium, Caruncle, Ovary and Fallopion tube (frozen and fixed)

A. 2x plastic topped EDTA vacutainers (clinical biochemistry)

2 x 50ml Falcon tubes EDTA.

Blood Sample: 100ml Clotted, 100ml Heparin & 200ml EDTA

150ml EDTA

Euthanase

Record: time of death, ear tag number, drugs administered and by whom.

(Sample CSF only if sampling can be completed within 15 mins of time of death)

Sample CSF: bijoux for archive, falcon tube for biochem (using sterile needle)

Exsanguinate

Histology tissues RIGHT; Transmission tissues LEFT

B.Remove head with Sterile Knife. Expose and sample:

BF, PLP1. Submandibular LN

BF 2. Submandibular salivary gland

BF, PLP4. Retropharyngeal LN

BF 5. Parotid salivary gland

FSa6.Facial nerve

BF 7.Masseter

BF8.Oral Mucosa

BF 9.Tongue (dorsum to include mucosa)

BF, PLP10.Tonsil

11.Pharynx

BF12.Nictitating membrane (L.-transmission, Lymphoid tissue, R.-histology, Whole eyelid)

C.Complete skinning of head , flame and remove calvarium:

13.Sample dura over cerebral hemisphere

Remove brain wearing sterile gloves and using sterile instruments, place on sterile foil.

3. Caudal medulla

14.Olfactory Frontal cortex

FSa Remove Obex block

Liq N2Remove a piece of Cerebellum, caudal medulla, frontal cortex and occipital cortex

Bisect the brain parasaggitally, Place the right half along with the obex in FSa

79. Rostral medulla (see diagram)

Dissect the left half of the brain as shown in diagram, remove frontal cortex, parietal

cortex, Occipital cortex, basal nuclei, thalamus, midbrain and brainstem, place in bags and

place on cardice.

If snap frozen tissue is not frozen within 90mins please note the time frozen on the PM

sheet

BF15.Pituitary (Bisect, left half – transmission, right half - histology)

BF16.Trigeminal ganglia

19.Conjunctiva

FSa17.Optic Nerve

18.Retina (left and right into separate bijoux)

FSa 20.Cranial cervical ganglia

BF 21.Respiratory epithelium (mid-turbinate)

D.Eviscerate carcass and sample:

BF,Liq N2 36.Spleen

BF 37.Liver

BF 38.Pancreas

39.Perirenal fat

BF 40.Kidney

BF41.Hepatic LN

BF 42.Adrenals

43.Urine

E.Drop out gut onto a clean table (Or covered floor) and sample:

BF, PLP44.Mesenteric LN

BF, PLP 45.Duodenum

BF, PLP46.Distal ileum (Complete)

47.Distal ileum, Peyer’s patches (separated from ileum)

Liq N2 Distal ileum 1g snap frozen

48.Distal ileum (excluding Peyer’s patches)

BF, PLP49.Jejunum

50.Jejunum Peyer’s patches (separated from ileum)

BF, PLP51.Spiral colon

BF52.Rumen

BF53.Omasum

BF54.Abomasum (pyloric)

BF55.Rectum

56.Faeces

F.Go back to the carcass and sample:

BF57.Aorta (abdominal)

BF58.Psoas

BF59.Diaphragm

FSa60.Phrenic nerve

BF 61.Lung (left caudal)

BF Lung (left cranial)

62.Pericardium

FSa67.Stellate ganglia

Remove heart

BF63.Heart - left ventricle

64.Heart valve (mitral)

BF Heart - right ventricle

BFHeart interventricular septum

BF65.Intercostal muscle

BF66.Bronchio- mediastinal LN

GFrom the trunk. Expose and sample:

BF22.Sternocephalicus

BF23.Cervical thymus

24.Trachea

BF25.Cranial oesophagus

BFThyroid

BF 34.Triceps

FSa 35.Radial nerve

BF 26.Skin from back

BF 27.Achilles tendon

BF28.Popliteal LN

BF 29.Semitendinosus

FSa 30.Sciatic nerve

BF31.Pre-femoral LN

BF 32.Longissimus dorsi

BF33.Prescapular LN

68.Bone marrow (sternum)

69.Bone (femur) - Put into sterile bag

H.Isolate and trim vertebral column Expose whole spinal cord and sample (See Diagram):

Cut cord for fixing at C7, T4, T9 and L2

70.DRG (mid – cervical) 3,4,5,6

71.C1 – C2

PLP C2 at level of nerve root (remove the DRG before fixing)

72.C2 – C3

73.DRG (mid – thoracic) 5,6,7,8

74.T9 – T10

PLP T10 at level of nerve root (remove the DRG before fixing)

75.T10 – T11

76.L2 – L3

PLP L3 at level of nerve root (remove the DRG before fixing)

77.L3 – L4

78.Cauda equina

FSaRemainder of Spinal cord [NB 8 Cervical, 13 Thoracic]

SE1736: Necropsy Protocol II

Check eartag to ensure correct animal has been selected before starting any procedure

Blood Sample: 2x plastic topped EDTA vacutainers (clinical biochemistry )

2 x 50ml Falcon tubes EDTA

150ml Clotted, 150ml Heparin & 200ml EDTA

150ml EDTA

Euthanase

Record time of death, ear tag number, drugs administered and by whom.

Exsanguinate

Commence sampling immediately. Selected tissues must be sampled and snap frozen within 45 minutes of death. Approx. 5g of each of below tissues in 0.5cm3 cubes (~ 10 pieces)  Liq N2

Head should be disarticulated immediately and the brain removed –operator 1

Abdomen should be opened up and the intestines tied off and placed on separate table (or Covered Floor) –operator 3 should then sample guts

Carcass must be sampled simultaneously by the 2nd operator

Operator 1Operator 2Operator 3

frontal cortexSpinal cord(C1-2)Liver

Brain stem caudal to the obexKidneySpleen

cerebellumAdrenalPancreas

hypothalamusThyroidDistal ileum

PituitaryTricepsSpiral colon

3rd eyelidSemitendinosusDistal Ileum Peyer’s patches

Bone marrow (sternum)Jejunum Peyer’s patches

After these tissues have been sampled, the usual procedure for sampling should be followed:

Histology tissues RIGHT; Transmission tissues LEFT

B.Expose and sample:

BF, PLP1. Submandibular LN

BF2. Submandibular salivary gland

BF, PLP4. Retropharyngeal LN

Fsa20.Cranial cervical ganglia

BF5. Parotid salivary gland

Fsa6.Facial nerve

BF7.Masseter

BF8.Oral Mucosa

BF9.Tongue (dorsum to include mucosa)

BF, PLP10.Tonsil

11.Pharynx

12.Nictitating membrane (Left –Transmission Lymphoid tissue)

19.Conjunctiva

Fsa17.Optic Nerve

18.Retina (Left and right into separate bijoux )

C.Complete Skinning of head , flame and remove calvarium:

13.Sample dura over cerebral hemisphere

3. Caudal medulla.

14.Frontal cortex (Left side )

Remove remainder of brain then remove Obex block

Bisect the brain parasaggitally, Place the right half along with the obex into Fsa

Liq N2Remove a piece of Cerebellum

Dissect the left half of the brain as shown in diagram 1.

79.Rostral medulla

Place remainder of brain in gripbags

Fsa16.Trigeminal ganglia

BF21.Respiratory epithelium (mid-turbinate)

D.Eviscerate carcass and sample:

BF36.Spleen

BF37.Liver

BF38.Pancreas

39.Perirenal fat

BF40.Kidney

BF41.Hepatic LN

42.Adrenals

43.Urine

E.Drop out gut onto a clean table (or covered floor) and sample:

BF, PLP 44.Mesenteric LN