Al l a f fected anima l s are o f the South Hampshi re breed of sheep and or i g i nate e i ther from the South I s land f lock described above , or from the exper imenta l f lock e s tabl i shed with sheep de rived from i t . Contro l anima l s wer e age -matched , appa rently heal thy sheep from the experimental flock and a 2 years old Rornney ewe . To date the combined number o f confirmed a f fected c a s e s from the origi n a l flock and the exper imenta l flock tota l s 1 6 , w i th the fol lowing case h i storie s , whi ch a re summa ri sed i n Tab l e 2 . I .
Cas e s l and 2 : Two 1 8 months o l d rams , described above on whi c h the original d iagno s i s o f c e ro i d- l ipofusc i no s i s was made .
Case 3 : A 1 5 month s o l d ewe hogge t , whi ch was born on the South I sl and prope r ty , but had been pastured on a property n e a r Palmerston North f o r 5 months . I n September , 1 9 7 6 the a n im a l
exhibited s i g n s o f bl indne s s and showed head tremors . She was ho sp i ta l i se d for c l i n i cal ob se rvation and euthanased in
November , 1 9 7 6 . Ti s sues were col lected for hi stolog i c a l and e l e c tronm i croscop i c a l examinat ion , a fter wh ich a d iagno s i s o f ceroid-l ipofu s c i nos i s wa s made .
Ca s e s 4 and 5 : Two ram l ambs 4 mon ths old had d i ed sudden l y on the South I s land property in December , 1976 . The owner stored the heads in a free z e r unti l they were sent to Massey Unive r s i ty , whe re l i ght and fluore scent microscopy o f brain indic ated a d i agno s i s o f ovine ceroid-l ipo fu sc i nos i s in both anima l s .
Case 6 : A ram from the South I s land property was hospi ta l i zed at Ma s sey Univers i ty i n Apr i l , 1 9 7 7 at 2 1 mon ths o f age . The anima l had shown s igns o f bl indness and exhib i te d head tremors for about 6 months . E l e c tronmic roscopy of a skin biopsy
i n d i cated a pre sumptive d ia gno s i s o f ovine c e ro i d- l ipofu sc inos i s , wh i ch wa s con f i rmed by further exten s ive h i s tological and
e l e ctronmic roscopical examinat ion a fter the anima l wa s euthanased in August , 1 97 7 , a t the age of 2 5 month s .
Case 7 : A 1 5 month o l d ram hogget from the South I s land prope rty had shown typ i c a l symptoms for about 4 months and a rrived a t Massey Univers ity in November , 1 9 7 7 . E l e ctron microscopy of a skin b iopsy sugge sted a d iagno s i s of ovine c eroi d - l ipo fu s c ino s i s . Thi s was confirmed by l ight and e le ctronmic ro scopy of other ti s sues after euthanas i a of the a n imal in Apr i l , 1 9 7 8 , at the age of 20 month s .
TABLE 2 . I
Case h i s to r i e s of South H3mpshi re sheep a f fected with c e ro id - l ipofuscino s i s
C a s e No . Sex 1 M a l e 2 Mal e 3 Fema le 4 Male 5 Ma l e 6 t-1al e 7 Male 8 Female 9 Ma le 10 Femal e 1 1 Mal e 1 2 Femal e 1 3 Ma l e 14 Fema l e 1 5 Fema l e 1 6 Mal e Or i g in Sth . I s land 1 1 1 1 1 1 1 1 11 1 1 1 1 11 11 Expt . f lock " 11 11 1 1 " 11 11 11 11 11 11 11 ,, 11 11 11 Age of onset (mon th s ) 14 14 13 15 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1
Present ing signs
B l i ndne s s , mu scle tremo r s Bl indness , mus c l e tremors B l indne s s , head tremors Found dead at 4 months Found dead at 4 months Bl indne s s , head tremor s Bl inde s s , head tremors B l indn e s s Bl indne s s , mus c l e tremo r s Bl indne s s Per inatal d e a th 11 1 1 B l indnes s 11 1 1 1 1
Ca se s 8 , 9 and 10 : The se anima l s were among the f i r s t crop o f l ambs produced in the experimenta l flock , in the spr i ng of 197 7 . F l uore s c ent , l ight a nd e l e ctronmic roscopy o f l iver and rectal biops i e s o f 36 l amb s , aged 4-5 months , reve a l e d these three anima l s to be su f fering from ceroid-l ipo fu s c inos i s . The two ewe l amb s ( c a s e s 8 and 1 0 ) exhibited c l inical symptoms at 1 1 months of age and the ram lamb at 12 months of age . Ca s e s 9 and 1 0
we re e lectively k i l l ed in October , 1 9 7 8 a n d the o r i g i n a l d iagno s i s was con f i rmed by fu rthe r hi stological examination . Case 8 gave bi rth to a ram l amb wh ich she suc c e s s fu l l y reared u n t i l she was euthana sed in Februa ry , 1 9 7 9 . The origina l di agno s i s was again con f i rmed by hi stolog i c a l examinat ion .
C a s e s 11 and 1 2 : A ma l e and female l amb in the experimental flock . Each was a member o f a set of twi n s and d i e d shortly
a fter b i r th during the 1 9 7 8 lambing s e a son . L i gh t and fluore scent m i c roscopy of brain showed that both were a f fe c te d with ceroid
l ipo fu s c i nos i s .
C a s e s 1 3 , 14 , 1 5 a nd 1 6 : Two ma les and two fema l e s out of a total o f 5 1 l ambs from the 1 9 7 8 lamb c rop o f the expe r imental f l ock were d iagnosed as being a f fected w i th c er o i d- l ipo fu s c ino s i s a t 4 - 5 months o f age . Diagnosis re s ted on the resu l t s of
f l uorescent micro scopy o f l iver and skin biopsy mate r i a l . A l l four animal s exhib ited c l in i c a l symptoms at l l months of a ge .
Haematological techniqu e s
l . Col lection o f bloodsampl e s : Blood samp l e s we re obta ined by j ugular venepuncture u s i ng 10 ml EDTA vacuum tube s . The samp l e s were used for e stimat ion o f the haemogram and for the prepara t ion o f fresh sme a r s .
2 . Haemogram dete rm ination : The routine e xamination i nvolved determinations of ce l l counts , di fferenti a l l eukocyte counts , haemoglob i n concentra t ion , packed c e l l volume , mean corpu scu l a r haemoglob i n concentra tion , total p rote i n , f ibrinogen , pl a sma protein to fibrinogen ratio and i cteric i ndex .
3 . Staining o f fresh smears for l eukocyte morphology :
Fre sh smears we re s ta ined with MacNeal ' s tetrachrome stain* a s fol lows :
( i ) Make up solution by d i s solving l . Sg o f s t a i n i n 5 00 ml of methyl a lcohol
( i i ) F i x by f looding smear for 3 m i n
( i i i ) Di lute w i th a s l ightly more than equal amount o f phosphate bu ffer (pH 6 . 8 ) mixing we l l . Leave for 6 min and wash o f f under tap . Dra i n dry
4 . Graham-Knol l techn ique for pe roxidase activi ty i n neutroph i l s : ( i ) Dry smea r i n a i r
( ii ) F i x i n forma ldehyde-alcohol ( l part o f 4 0% forma l in and 9 parts of 96% a lcoho l )
( i i i ) Rin se in wa te r
( iv ) Pour o n benzidine reagent and leave for 0 . 5 - 5 . 0 min a t 3 0 s interva l s
( v ) Pour off , rinse and d ry
( v i ) Sta i n with d i lute Giemsa ( 1 0 drops stock sol ution i n 10 ml d i s t i l l ed wate r ) for about 45 min
(vi i ) Rin s e and dry
Benzidine reagent