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Effects of different doses of dog-derived Sarcocystis sporocysts on growth rate and haematocrit in lambs
Veterinary Parasitology, 21 (1986) 21--24 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
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EFFECTS OF DIFFERENT DOSES OF DOG-DERIVED SARCOCYSTIS S P O R O C Y S T S ON G R O W T H R A T E A N D H A E M A T O C R I T IN LAMBS
B.L. MUNDAY
Mount Pleasant Laboratories, P.O. Box 46, Launceston South, Tasmania 7249 (Australia) (Accepted for publication 17 July 1985) ABSTRACT Munday B.L., 1986. Effects of different doses of dog-derived Sarcocystis sporocysts on growth rate and haematocrit in lambs. Vet. Parasitol., 21: 21--24. Four-week-old lambs at pasture were dosed with dog-derived Sarcocystis sporocysts. No difference in growth rates was apparent at a dose of 1 × 103 sporocysts per lamb. The minimum dose required to depress growth rate was 2.5 × 10 ~ sporocysts per lamb: at 4 weeks post-inoculation (w.p.i.) the weight gain of lambs infected with this dose was 0.6 kg less than the non-infected controls (P < 0.05). At 11 w.p.i, the difference was 0.7 kg, but this was not significant because of the greater average body weights of both groups. Lambs given 5 × 103 sporocysts showed significant depression of weight gain at both 4 and 11 w.p.i. Haematocrit levels at 4--5 weeks post-inoculation were depressed by doses as low 1 × 103 sporocysts.
INTRODUCTION M o d e s t d o s e s (5 × 1 0 3 - - 2 . 5 × 104) o f d o g - d e r i v e d s p o r o c y s t s o f Sarcocystis tenella and Sarcocystis sp. h a v e b e e n s h o w n t o s i g n i f i c a n t l y d e p r e s s t h e g r o w t h r a t e a n d h a e m a t o c r i t levels o f y o u n g l a m b s ( M u n d a y , 1 9 7 9 , 1 9 8 4 ; Erber and Burgkart, 1981). H o w e v e r , t h e l o w e s t p a t h o g e n i c d o s e o f s p o r o c y s t s was n o t d e t e r m i n e d in a n y o f t h e s e p r e v i o u s l y - r e p o r t e d trials. T h i s s t u d y was u n d e r t a k e n in a n attempt to define the minimum pathogenic dose of sporocysts, even though a n y findings w o u l d o n l y strictly relate to the p a r t i c u l a r c o n d i t i o n s of these trials. MATERIALS AND METHODS T h e t r i a l s w e r e c o n d u c t e d o n T h r e e H u m m o c k I s l a n d w h i c h is s i t u a t e d i n Bass S t r a i t o f f t h e n o r t h w e s t t i p o f T a s m a n i a . T h i s i s l a n d w a s c h o s e n b e c a u s e d o g s h a v e n o t b e e n p e r m i t t e d t o l a n d in t h e i n h a b i t e d a r e a f o r a t least 15 y e a r s . A l s o , t h e trials r e p o r t e d b y M u n d a y ( 1 9 7 9 , 1 9 8 4 ) w e r e p e r f o r m e d o n t h e s a m e area.
0304-4017/86/$03.50
© 1986 Elsevier Science Publishers B.V.
22
Trial 1 T h e e x p e r i m e n t a l animals were 64 c r o s s b r e d l a m b s w h i c h were a p p r o x i m a t e l y 1 m o n t h o f age w h e n the trial c o m m e n c e d . On 9 N o v e m b e r 1983 the l a m b s w e r e weighed and bled and 32 of t h e m w e r e d o s e d orally w i t h 1 × 10 s d o g - d e r i v e d Sarcocystis s p o r o c y s t s . These l a m b s (and their d a m s ) were grazed s e p a r a t e l y f r o m t h e r e m a i n i n g 32 l a m b s f o r 10 days. At 5 and 11 w e e k s p o s t - i n o c u l a t i o n (w.p.i.) the l a m b s were w e i g h e d and bled, and at 11 w.p.i, t h e i r tails were r e m o v e d f o r e x a m i n a t i o n f o r s a r c o c y s t s .
Trial 2 T h e e x p e r i m e n t a l animals were 106 crossbred l a m b s which were a p p r o x i m a t e l y o n e m o n t h o f age w h e n the trial c o m m e n c e d . O n 2 O c t o b e r 1984 the l a m b s were w e i g h e d and bled. T h i r t y - f i v e a n i m a l s were each d o s e d orally with 2.5 X 10 s d o g - d e r i v e d s p o r o c y s t s and 37 were each d o s e d w i t h 5 X 10 ~ s p o r o c y s t s . T h e s e 72 a n i m a l s (and t h e i r d a m s ) w e r e grazed s e p a r a t e l y f r o m t h e r e m a i n i n g 34 f o r 10 days. At 4 a n d 11 w.p.i, the l a m b s w e r e weighed and bled, a n d at 11 w.p.i, t h e i r tails w e r e r e m o v e d for e x a m i n a t i o n f o r sarcocysts. S p o r o c y s t s f o r these trials were supplied b y Dr G. F o r d of the S o u t h A u s t r a l i a n D e p a r t m e n t of A g r i c u l t u r e and h a d b e e n r e c o v e r e d f r o m the faeces o f specific p a t h o g e n - f r e e dogs w h i c h h a d b e e n fed on m u t t o n f r o m the S o u t h Australian Meat C o r p o r a t i o n , Adelaide. S p o r o c y s t s were s e p a r a t e d b y f l o t a t i o n o n s a t u r a t e d sugar s o l u t i o n a n d s t o r e d in a n t i b i o t i c s o l u t i o n ( 1 0 0 units penicillin, 100 pg s t r e p t o m y c i n and 50 pg a m p h o t e r i c i n B ml -t ) for n o t m o r e t h a n 30 d a y s p r i o r to i n o c u l a t i o n . Muscle tissues f r o m the l a m b s ' tails w e r e p r o c e s s e d by r o u t i n e p r o c e d u r e s f o r histological e x a m i n a t i o n . Analysis of variance was used to test for significant d i f f e r e n c e s b e t w e e n the g r o u p s w i t h regard to w e i g h t gain a n d h a e m a t o c r i t . RESULTS Mean w e i g h t gains are s h o w n in T a b l e I. Statistical analysis i n d i c a t e d t h a t a d o s e of 1 X 103 s p o r o c y s t s per l a m b did n o t p r o d u c e a n y significant effect. T h e smallest p a t h o g e n i c dose was 2.5 × 103 s p o r o c y s t s per l a m b : this prod u c e d a significant d e p r e s s i o n of weight gain 4 w.p.i;, b u t n o t at 11 w.p.i. A dose o f 5 X l 0 s s p o r o c y s t s per l a m b p r o d u c e d a significant d e p r e s s i o n o f w e i g h t gain at b o t h 4 and 11 w.p.i. H a e m a t o c r i t level p r o v e d t o be a m o r e sensitive m e a s u r e o f t h e e f f e c t o f Sarcocystis i n f e c t i o n (Table II). All d o s e levels p r o d u c e d a significant depression at 4--5 w.p.i., b u t o n l y a d o s e of 5 X 10 ~ s p o r o c y s t s per l a m b s p r o d u c e d a significant e f f e c t at 11 w.p.i. M a t u r e s a r c o c y s t s w e r e f o u n d in the c o c c y g e a l muscles of the d o s e d , b u t n o t the u n d o s e d lambs.
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TABLE I M e a n w e i g h t s o f l a m b s in Trial no. (year}
1 (1983/1984) 2 (1984)
Sarcocystis trials
Treatment (sporocysts/animal)
0 1 x 103 0 2.5 x 103 5 :~ 103
Weight preinoculation (kg) 16.9 18.2 12.7 12.3 12.5
Weight p o s t - i n o c u l a t i o n (kg) at w e e k 4
5
11
25.3 27.0
30.2 31.1 27.7 a 26.6 25.8 a
20.0 ab 19.0 a 19.3 b
V a l u e s w i t h t h e s a m e letter n o t a t i o n are s i g n i f i c a n t l y d i f f e r e n t (P <: 0.05 ).
T A B L E II M e a n h a e m a t o c r i t s (%) o f l a m b s in Trial no. (year)
Sarcocystis trials
Treatment (sporocysts/animal)
Preinoculation
P o s t - i n o c u l a t i o n {weeks) 4
1{1983/1984) 2 (1984)
0 1 x 103 0 2.5 x 103 5 x 10 ~
35.6 32.6 33.6 29.7 30.1
5
11
36.2 a 32.4 a
37.6 34.8 39.2 a 35.5 34.7 a
37.0 a 31,2 a 29.3 a
V a l u e s w i t h t h e s a m e l e t t e r n o t a t i o n are s i g n i f i c a n t l y d i f f e r e n t (P < 0 . 0 0 1 ) .
DISCUSSION
Even though the results reported here and previously (Munday, 1979, 1984) were obtained in different years, t hey are comparable in that the same flock o f sheep and area of pasture were used for each trial. These results may also form the basis for more general prognostications concerning the pathogenicity of dog-derived Sarcocystis spp. for sheep, but would need qualification depending upon the envi r onm ent and genetic background and age of the sheep. In Australia, fat lambs are rarely killed before 12 weeks of age and therefore single doses < 5 X 103 sporocysts per lamb are not likely to be of e c o n o mic importsnce. It is more likely that lambs will be subject to "trickle' infection co mmen c i ng when t h e y start to graze and no information is available on the effects of this t y p e of challenge. The degrees o f anaemia p r o d u c e d by doses ofSarcocystis sporocysts as high as 1 X 104 per lamb (Munday, 1979) are not significant in themselves. How-
24 ever, if l a m b s w e r e suffering f r o m c o n c u r r e n t a n a e m i a d u e to h a e m o n c h o s i s , e p e r y t h r o z o o n o s i s or o t h e r causes, the additive e f f e c t s c o u t d be very deleterious, p o s s i b l y causing d e a t h . In the light o f D u b e y ' s ( 1 9 8 1 ) w o r k w i t h goats in which it was s h o w n t h a t 1 × 102- 1 × 103 S. capracanis s p o r o c y s t s w e r e suitable, n o n - p a t h o g e n i c i m m u n i z i n g doses, it is p r o b a b l e t h a t e q u i v a l e n t doses w o u l d be effective i m m u n o g e n s in lambs. I n d e e d , F o r d ( 1 9 8 5 ) has s h o w n t h a t a dose of 5 × 10-" s p o r o c y s t s given to s h e e p a m e l i o r a t e d t h e e f f e c t s of a s u b s e q u e n t challenge with 5 × l 0 w d o g - d e r i v e d Sarcocystis s p o r o c y s t s . In o r d e r to a p p l y these findings to t h e field s i t u a t i o n it will be necessary to establish t h e r e l a t i o n s h i p (if a n y ) b e t w e e n single dose c h a l l e n g e s and t h e t y p e o f ' t r i c k l e ' i n f e c t i o n p r e s u m e d to o c c u r u n d e r n o r m a l grazing situations. ACKNOWLEDGEMENTS M a n y officers o f the T a s m a n i a n D e p a r t m e n t o f A g r i c u l t u r e assisted with this w o r k . Mr. D. G a t e n b y o f the T a s m a n i a n N a t i o n a l Parks and Wildlife Service c o - o r d i n a t e d the m a n a g e m e n t o f the sheep. Sarcocystis s p o r o c y s t s were kindly p r o v i d e d b y Dr. G. F o r d o f t h e S o u t h Australian D e p a r t m e n t o f A g r i c u l t u r e . Finm]cial assistance was p r o v i d e d b y the Australian M e a t Research Committee. REFERENCES Dubey, J.P., 1981. Development of immunity to sarcocystosis in dairy goats. Am. J. Vet. Res., 42: 800--804. Erber, M. and Burgkart, M., 1981. Wirtschaftliche Verluste durch Sarkosporidiose (Sarcocyslis ovicanis und S. spec.) bei der Mast yon Schaflammern. Prakt. Tierarztl., 62: -122--427. Ford, G.E., 1985. Immunity of sheep to homologous challenge with dog-borne Sarcocystis species following varying levels of prior exposure. Int. J. Parasitol., 15: 629--634. Munday, B.L., 1979. The effect of Sareoeystis ovicanis on growth and haematoerit in Iambs. Vet. Parasitol., 5: 129--135. Munday, B.L., 1984. The effect of Sarcoeystis tetzella on wool growth in sheep. Vet. Parasitol., 15 : 91--94.
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EFFECTS OF DIFFERENT DOSES OF DOG-DERIVED SARCOCYSTIS S P O R O C Y S T S ON G R O W T H R A T E A N D H A E M A T O C R I T IN LAMBS
B.L. MUNDAY
Mount Pleasant Laboratories, P.O. Box 46, Launceston South, Tasmania 7249 (Australia) (Accepted for publication 17 July 1985) ABSTRACT Munday B.L., 1986. Effects of different doses of dog-derived Sarcocystis sporocysts on growth rate and haematocrit in lambs. Vet. Parasitol., 21: 21--24. Four-week-old lambs at pasture were dosed with dog-derived Sarcocystis sporocysts. No difference in growth rates was apparent at a dose of 1 × 103 sporocysts per lamb. The minimum dose required to depress growth rate was 2.5 × 10 ~ sporocysts per lamb: at 4 weeks post-inoculation (w.p.i.) the weight gain of lambs infected with this dose was 0.6 kg less than the non-infected controls (P < 0.05). At 11 w.p.i, the difference was 0.7 kg, but this was not significant because of the greater average body weights of both groups. Lambs given 5 × 103 sporocysts showed significant depression of weight gain at both 4 and 11 w.p.i. Haematocrit levels at 4--5 weeks post-inoculation were depressed by doses as low 1 × 103 sporocysts.
INTRODUCTION M o d e s t d o s e s (5 × 1 0 3 - - 2 . 5 × 104) o f d o g - d e r i v e d s p o r o c y s t s o f Sarcocystis tenella and Sarcocystis sp. h a v e b e e n s h o w n t o s i g n i f i c a n t l y d e p r e s s t h e g r o w t h r a t e a n d h a e m a t o c r i t levels o f y o u n g l a m b s ( M u n d a y , 1 9 7 9 , 1 9 8 4 ; Erber and Burgkart, 1981). H o w e v e r , t h e l o w e s t p a t h o g e n i c d o s e o f s p o r o c y s t s was n o t d e t e r m i n e d in a n y o f t h e s e p r e v i o u s l y - r e p o r t e d trials. T h i s s t u d y was u n d e r t a k e n in a n attempt to define the minimum pathogenic dose of sporocysts, even though a n y findings w o u l d o n l y strictly relate to the p a r t i c u l a r c o n d i t i o n s of these trials. MATERIALS AND METHODS T h e t r i a l s w e r e c o n d u c t e d o n T h r e e H u m m o c k I s l a n d w h i c h is s i t u a t e d i n Bass S t r a i t o f f t h e n o r t h w e s t t i p o f T a s m a n i a . T h i s i s l a n d w a s c h o s e n b e c a u s e d o g s h a v e n o t b e e n p e r m i t t e d t o l a n d in t h e i n h a b i t e d a r e a f o r a t least 15 y e a r s . A l s o , t h e trials r e p o r t e d b y M u n d a y ( 1 9 7 9 , 1 9 8 4 ) w e r e p e r f o r m e d o n t h e s a m e area.
0304-4017/86/$03.50
© 1986 Elsevier Science Publishers B.V.
22
Trial 1 T h e e x p e r i m e n t a l animals were 64 c r o s s b r e d l a m b s w h i c h were a p p r o x i m a t e l y 1 m o n t h o f age w h e n the trial c o m m e n c e d . On 9 N o v e m b e r 1983 the l a m b s w e r e weighed and bled and 32 of t h e m w e r e d o s e d orally w i t h 1 × 10 s d o g - d e r i v e d Sarcocystis s p o r o c y s t s . These l a m b s (and their d a m s ) were grazed s e p a r a t e l y f r o m t h e r e m a i n i n g 32 l a m b s f o r 10 days. At 5 and 11 w e e k s p o s t - i n o c u l a t i o n (w.p.i.) the l a m b s were w e i g h e d and bled, and at 11 w.p.i, t h e i r tails were r e m o v e d f o r e x a m i n a t i o n f o r s a r c o c y s t s .
Trial 2 T h e e x p e r i m e n t a l animals were 106 crossbred l a m b s which were a p p r o x i m a t e l y o n e m o n t h o f age w h e n the trial c o m m e n c e d . O n 2 O c t o b e r 1984 the l a m b s were w e i g h e d and bled. T h i r t y - f i v e a n i m a l s were each d o s e d orally with 2.5 X 10 s d o g - d e r i v e d s p o r o c y s t s and 37 were each d o s e d w i t h 5 X 10 ~ s p o r o c y s t s . T h e s e 72 a n i m a l s (and t h e i r d a m s ) w e r e grazed s e p a r a t e l y f r o m t h e r e m a i n i n g 34 f o r 10 days. At 4 a n d 11 w.p.i, the l a m b s w e r e weighed and bled, a n d at 11 w.p.i, t h e i r tails w e r e r e m o v e d for e x a m i n a t i o n f o r sarcocysts. S p o r o c y s t s f o r these trials were supplied b y Dr G. F o r d of the S o u t h A u s t r a l i a n D e p a r t m e n t of A g r i c u l t u r e and h a d b e e n r e c o v e r e d f r o m the faeces o f specific p a t h o g e n - f r e e dogs w h i c h h a d b e e n fed on m u t t o n f r o m the S o u t h Australian Meat C o r p o r a t i o n , Adelaide. S p o r o c y s t s were s e p a r a t e d b y f l o t a t i o n o n s a t u r a t e d sugar s o l u t i o n a n d s t o r e d in a n t i b i o t i c s o l u t i o n ( 1 0 0 units penicillin, 100 pg s t r e p t o m y c i n and 50 pg a m p h o t e r i c i n B ml -t ) for n o t m o r e t h a n 30 d a y s p r i o r to i n o c u l a t i o n . Muscle tissues f r o m the l a m b s ' tails w e r e p r o c e s s e d by r o u t i n e p r o c e d u r e s f o r histological e x a m i n a t i o n . Analysis of variance was used to test for significant d i f f e r e n c e s b e t w e e n the g r o u p s w i t h regard to w e i g h t gain a n d h a e m a t o c r i t . RESULTS Mean w e i g h t gains are s h o w n in T a b l e I. Statistical analysis i n d i c a t e d t h a t a d o s e of 1 X 103 s p o r o c y s t s per l a m b did n o t p r o d u c e a n y significant effect. T h e smallest p a t h o g e n i c dose was 2.5 × 103 s p o r o c y s t s per l a m b : this prod u c e d a significant d e p r e s s i o n of weight gain 4 w.p.i;, b u t n o t at 11 w.p.i. A dose o f 5 X l 0 s s p o r o c y s t s per l a m b p r o d u c e d a significant d e p r e s s i o n o f w e i g h t gain at b o t h 4 and 11 w.p.i. H a e m a t o c r i t level p r o v e d t o be a m o r e sensitive m e a s u r e o f t h e e f f e c t o f Sarcocystis i n f e c t i o n (Table II). All d o s e levels p r o d u c e d a significant depression at 4--5 w.p.i., b u t o n l y a d o s e of 5 X 10 ~ s p o r o c y s t s per l a m b s p r o d u c e d a significant e f f e c t at 11 w.p.i. M a t u r e s a r c o c y s t s w e r e f o u n d in the c o c c y g e a l muscles of the d o s e d , b u t n o t the u n d o s e d lambs.
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TABLE I M e a n w e i g h t s o f l a m b s in Trial no. (year}
1 (1983/1984) 2 (1984)
Sarcocystis trials
Treatment (sporocysts/animal)
0 1 x 103 0 2.5 x 103 5 :~ 103
Weight preinoculation (kg) 16.9 18.2 12.7 12.3 12.5
Weight p o s t - i n o c u l a t i o n (kg) at w e e k 4
5
11
25.3 27.0
30.2 31.1 27.7 a 26.6 25.8 a
20.0 ab 19.0 a 19.3 b
V a l u e s w i t h t h e s a m e letter n o t a t i o n are s i g n i f i c a n t l y d i f f e r e n t (P <: 0.05 ).
T A B L E II M e a n h a e m a t o c r i t s (%) o f l a m b s in Trial no. (year)
Sarcocystis trials
Treatment (sporocysts/animal)
Preinoculation
P o s t - i n o c u l a t i o n {weeks) 4
1{1983/1984) 2 (1984)
0 1 x 103 0 2.5 x 103 5 x 10 ~
35.6 32.6 33.6 29.7 30.1
5
11
36.2 a 32.4 a
37.6 34.8 39.2 a 35.5 34.7 a
37.0 a 31,2 a 29.3 a
V a l u e s w i t h t h e s a m e l e t t e r n o t a t i o n are s i g n i f i c a n t l y d i f f e r e n t (P < 0 . 0 0 1 ) .
DISCUSSION
Even though the results reported here and previously (Munday, 1979, 1984) were obtained in different years, t hey are comparable in that the same flock o f sheep and area of pasture were used for each trial. These results may also form the basis for more general prognostications concerning the pathogenicity of dog-derived Sarcocystis spp. for sheep, but would need qualification depending upon the envi r onm ent and genetic background and age of the sheep. In Australia, fat lambs are rarely killed before 12 weeks of age and therefore single doses < 5 X 103 sporocysts per lamb are not likely to be of e c o n o mic importsnce. It is more likely that lambs will be subject to "trickle' infection co mmen c i ng when t h e y start to graze and no information is available on the effects of this t y p e of challenge. The degrees o f anaemia p r o d u c e d by doses ofSarcocystis sporocysts as high as 1 X 104 per lamb (Munday, 1979) are not significant in themselves. How-
24 ever, if l a m b s w e r e suffering f r o m c o n c u r r e n t a n a e m i a d u e to h a e m o n c h o s i s , e p e r y t h r o z o o n o s i s or o t h e r causes, the additive e f f e c t s c o u t d be very deleterious, p o s s i b l y causing d e a t h . In the light o f D u b e y ' s ( 1 9 8 1 ) w o r k w i t h goats in which it was s h o w n t h a t 1 × 102- 1 × 103 S. capracanis s p o r o c y s t s w e r e suitable, n o n - p a t h o g e n i c i m m u n i z i n g doses, it is p r o b a b l e t h a t e q u i v a l e n t doses w o u l d be effective i m m u n o g e n s in lambs. I n d e e d , F o r d ( 1 9 8 5 ) has s h o w n t h a t a dose of 5 × 10-" s p o r o c y s t s given to s h e e p a m e l i o r a t e d t h e e f f e c t s of a s u b s e q u e n t challenge with 5 × l 0 w d o g - d e r i v e d Sarcocystis s p o r o c y s t s . In o r d e r to a p p l y these findings to t h e field s i t u a t i o n it will be necessary to establish t h e r e l a t i o n s h i p (if a n y ) b e t w e e n single dose c h a l l e n g e s and t h e t y p e o f ' t r i c k l e ' i n f e c t i o n p r e s u m e d to o c c u r u n d e r n o r m a l grazing situations. ACKNOWLEDGEMENTS M a n y officers o f the T a s m a n i a n D e p a r t m e n t o f A g r i c u l t u r e assisted with this w o r k . Mr. D. G a t e n b y o f the T a s m a n i a n N a t i o n a l Parks and Wildlife Service c o - o r d i n a t e d the m a n a g e m e n t o f the sheep. Sarcocystis s p o r o c y s t s were kindly p r o v i d e d b y Dr. G. F o r d o f t h e S o u t h Australian D e p a r t m e n t o f A g r i c u l t u r e . Finm]cial assistance was p r o v i d e d b y the Australian M e a t Research Committee. REFERENCES Dubey, J.P., 1981. Development of immunity to sarcocystosis in dairy goats. Am. J. Vet. Res., 42: 800--804. Erber, M. and Burgkart, M., 1981. Wirtschaftliche Verluste durch Sarkosporidiose (Sarcocyslis ovicanis und S. spec.) bei der Mast yon Schaflammern. Prakt. Tierarztl., 62: -122--427. Ford, G.E., 1985. Immunity of sheep to homologous challenge with dog-borne Sarcocystis species following varying levels of prior exposure. Int. J. Parasitol., 15: 629--634. Munday, B.L., 1979. The effect of Sareoeystis ovicanis on growth and haematoerit in Iambs. Vet. Parasitol., 5: 129--135. Munday, B.L., 1984. The effect of Sarcoeystis tetzella on wool growth in sheep. Vet. Parasitol., 15 : 91--94.