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Resistance to Eimeria bovis produced after chemotherapy of experimental infections in calves
Veterinary Parasitology, 9 (1982) 171--177 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
RESISTANCE TO EIMERIA BOVIS PRODUCED OF EXPERIMENTAL INFECTIONS IN CALVES
AFTER
171
CHEMOTHERAPY
P.H.G. STOCKDALE and ANNE SHEARD Department o f Veterinary Microbiology, University o f Saskatchewan, Saskatoon, Saskatche wan S 7N 0 WO i(Canada)
G.B. T I F F I N Animal Disease Research Institute, Box 640, Lethbridge, Alberta TIJ 3Z4 (Canada)
(Accepted for publication 13 August 1981 )
ABSTRACT
Stockdale, P.H.G., Sheard, A. and Tiffin, G.B., 1982. Resistance to Eimeria bovis produced after chemotherapy of experimental infections in calves. Vet. Parasitol., 9: 171--177. Eighteen Holstein-Friesian four week old calves were inoculated with 5 x 10 s sporocysts of Eimeria boris by stomach tube. The calves were divided into three equal groups. The first group was treated with monensin, 1 mg kg-' body weight daily, from the 10th-20th day inclusive after infection; the second group was treated with amprolium, 10 mg kg - ' body weight daily, for the same period of time and the third group acted as infected controls. Both drugs were for the most part effective in preventing clinical signs, in suppressing reduced rates of weight and in reducing oocyst production. The calves of all three groups were resistant to the second infection given 35 days after the first infection with E. bovis as measured by rates of weight gain, fecal oocyst output and lack of clinical signs. INTRODUCTION L e v i n e a n d I v e n s ( 1 9 7 0 ) d e s c r i b e d E i m e r i a z u e r n i i a n d E. b o v i s as b e i n g t h e t w o m o s t p a t h o g e n i c s p e c i e s o f c o c c i d i a in c a t t l e . I n e a r l i e r w o r k t h e e f f e c t s o f t r e a t m e n t o f E. z u e r n i i i n f e c t i o n s in c a l v e s w i t h m o n e n s i n a n d amprolium and the development of resistance to challenge infection of the calves were described (Stockdale and Yates, 1978). It was decided to do the f o l l o w i n g e x p e r i m e n t o n E. b o v i s t o see i f a s i m i l a r i n f e c t i o n a n d t r e a t m e n t r e g i m e n w i t h t h i s l a t t e r p r o t o z o a n w o u l d p r o d u c e s i m i l a r r e s u l t s t o t h o s e rep o r t e d f o r E. z u e r n i i , as a n y p r a c t i c a l p r o p h y l a c t i c t r e a t m e n t f o r c o c c i d i o s i s in c a t t l e w o u l d n e e d t o b e d i r e c t e d a g a i n s t b o t h o f t h e a b o v e o r g a n i s m s . MATERIALS AND METHODS Eighteen Holstein-Friesian calves were raised under "minimal coccidiosis" c o n d i t i o n s as d e s c r i b e d e a r l i e r ( S t o c k d a l e a n d Y a t e s , 1 9 7 8 ) . T h e c a l v e s , a p p r o x i m a t e l y f o u r w e e k s o f a g e , w e r e i n f e c t e d w i t h 5 X 1 0 s s p o r o c y s t s o f E. b o v i s 0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
172 suspended in saline via a stomach tube. All calves were given approximately 300 ml o f r u men fluid from adult cattle five days after infection, in an att e m p t to provide conventional enteric flora and fauna. This was provided because the calves had been raised under "minimal disease" conditions and had had no c o n t a c t with adult cattle, thus they had n o t acquired a " n o r m a l " intestinal biota. There was some slight risk that coccidia, ot her than the dose given, were added with the rumen fluid but this risk was taken as it was considered to be i m p o r t a n t to provide the calves with rum en organisms that would allow t h e m to make more use of the alfalfa pellets and dairy concentrate with which t hey were being fed. The calves were injected intramuscularly with 40 mg of de xa m et has one (Dexagen 5 or 2, Rogar/STB, L o n d o n , Ontario) on Days 13, 14 and 15 after infection (Stockdale and Niilo, 1976). This was used, to be consistent with the E. zuernii infections described earlier (Stockdale and Yates, 1978), as it was desirable to examine E. boris under similar conditions. Calves were infected in replicates of three animals. All were bull calves, with the ex cep tion of one free-martin heifer calf. The calves were given drugs f r o m Day 10 to Day 20 inclusive post-infection (PI), and bot h drugs were fed so that the daily dose was divided into two parts and fed in milk replacer. The first calf of each replicate was given 1.0 mg kg -1 b o d y weight of monensin (Coban, Elanco Ltd., IN), the second calf was given 10 mg kg -1 of amprolium (Amprovine, Merck, NY) and the third calf remained as an untreated, infected control. There were six calves in each t r e a t m e n t and c o n tr o l group. Calves were weighed twice weekly and from Day 13 all feces passed were collected at 24 h intervals. The total daily fecal o u t p u t of oocysts per calf was estimated as previously described (Stockdale, 1976) until Day 30 after infection. Calves were reinfected on Day 35 (PI) with 5 X 105 sporocysts of E. bovis and the various required parameters measured as in the first infection. Fecal o o c y s t o u t p u t f r om this second infection in the calves was estim a t e d from Day 13--Day 25 (49--60 days after the first infection). The calves were n o t treated with any drugs during the second infection. The oocysts of E. bovis used as a source of sporocysts t h r o u g h o u t the e x p e r i m e n t were fr om a single c o m m o n pool and, as the work was done with successive replicates, a following replicate acted as a control for oocyst infectivity for each challenge infection. RESULTS All the calves survived the experimental period but all calves in the last three replicates developed concur r ent enzootic pneumonia. As this appeared to affect all the calves, it was assumed that the comparisons made between the coccidial infections were still valid. The control calves had clinical coccidiosis during the first infection. Their feces became diarrhoeic and later contained blood, mucus, large sheets of
173 m u c o s a a n d fibrin casts. T h e r e was d e h y d r a t i o n a n d t e n e s m u s in the m o s t severely a f f e c t e d c o n t r o l calves a n d t h e s e signs o c c u r r e d at a p p r o x i m a t e l y the s a m e t i m e as p e a k o o c y s t p r o d u c t i o n . T h e clinical signs w e r e m u c h red u c e d in t h e calves t r e a t e d w i t h a m p r o l i u m . O n l y t w o o f these calves h a d mild d i a r r h o e a f r o m D a y 2 0 - - D a y 22 a f t e r i n f e c t i o n . H o w e v e r t w o calves (calves 5 and 6) o f the g r o u p t r e a t e d w i t h m o n e n s i n h a d clinical disease. T h e s e calves h a d d i a r r h o e a a n d d y s e n t e r y and t h e i r feces c o n t a i n e d casts o f epithelial cells, b u t n o n e o f t h e s e signs were as severe as t h o s e seen in t h e u n t r e a t e d c o n t r o l calves. All calves' feces w e r e o f n o r m a l c o n s i s t e n c y b y D a y 30 a f t e r i n f e c t i o n . In t h e first i n f e c t i o n , p e a k o o c y s t p r o d u c t i o n f o r E. b o v i s o c c u r r e d on D a y 21 f o r t h e calves o f t h e m o n e n s i n and c o n t r o l g r o u p s and on D a y 25 f o r t h e calves o f t h e g r o u p given a m p r o l i u m . In t h e s e c o n d i n f e c t i o n , w i t h o u t t r e a t m e n t , t h e p e a k o f o o c y s t p r o d u c t i o n was on D a y 21 for all t h r e e groups. F r o m T a b l e I it c a n be seen t h a t in t h e first i n f e c t i o n b o t h drugs d e p r e s s e d o o c y s t o u t p u t f r o m t h e calves as c o m p a r e d t o t h a t o f t h e c o n t r o l calves. T h e r e d u c t i o n in o u t p u t was statistically significantly d i f f e r e n t using t h e MannW h i t n e y U t e s t ( G i b b o n s , 1 9 7 6 ) . F r o m T a b l e II it is a p p a r e n t t h a t t h e r e was little d i f f e r e n c e in m e a n t o t a l fecal o o c y s t o u t p u t o f t h e t h r e e g r o u p s o f calves d u r i n g t h e s e c o n d i n f e c t i o n . T h e r e was n o statistically significant d i f f e r e n c e b e t w e e n t h e s e m e a n values using t h e M a n n - W h i t n e y U test. TABLE I Fecal oocyst output of calves infected with E. bovis of the first infection Replicate
1 2 3 4 5 6 Mean
Treatment Monensin
Amprolium
Control
3.961 94.11 4.22 92.42 229.42 308.04 122.032
2.05 60.15 0.00 0.11 51.46 7.53 20.222
1910.59 247.93 113.92 184.00 430.11 407.15 548.952
1 × 10 60ocysts. 2 Statistically significant (Mann-Whitney U test). T h e p e r c e n t a g e w e i g h t gains f o r the t h r e e g r o u p s o f calves f r o m D a y 1 3 D a y 30 PI f o r the first i n f e c t i o n are given in Fig. 1. T h e lines o f b e s t fit w e r e d r a w n t o t h e values b y h a n d . All t h r e e lines are similar in c h a r a c t e r , t h a t is sigmoid in shape. T h u s it a p p e a r s t h a t in t h e c o n t r o l g r o u p t h e r e was a m e a n loss in w e i g h t f r o m D a y 18 t o D a y 22 a f t e r i n f e c t i o n , w h e r e a s in t h e t w o d r u g t r e a t e d g r o u p s t h e r e was n o increase in w e i g h t b e t w e e n D a y 18 a n d D a y 24 a f t e r i n f e c t i o n . T h e d i f f e r e n c e s in w e i g h t c h a n g e b e t w e e n the t h r e e g r o u p s
174 TABLE II Fecal oocyst output of calves infected with E. bovis of the second infection Replicate
Treatment Monensin
Amprolium
Control
1 2 3 4
1.101 0.00 0.00 12.02
17.18 1.17 0.00 0.00
0.00 0.00 0.08 0.00
5
+re 2
+re
+re
6
0.00
0.00
0.00
Mean
2.103
3.063
0.023
1× 106 Oocysts. 2 Only detected on flotation. 3 Not statistically significant (Mann-Whitney U test.).
20
©
•
a _c a~
©
OControluntreatedcalves
•
:
J
Amprolium treated calves •
•
Monensin
14
16
18
20
22
treatedcalves
24
26
28
•
30
OaysAfterInfection Fig. 1. Mean weight gains of calves after the first infection with Eimeria bovis. in t h e f i r s t i n f e c t i o n w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t ( G u n t h e r , 1 9 6 4 , F t e s t ) . T h e p e r c e n t a g e w e i g h t g a i n s f o r t h e t h r e e g r o u p s o f c a l v e s in t h e s e c o n d i n f e c t i o n a r e g i v e n in F i g . 2. T h e t h r e e g r o u p s o f c a l v e s g a i n e d weight at similar rates which were not statistically significantly different (F test).
175
60
J 5O .= ~3 .c O
O
40
~
O
© Control untreated calves
O
Amprolium treated calves •
30
Monensin treated calves
14
16
18
20
22
24
26
28
•
30
Days After Infection
Fig. 2. Mean weight gains of calves after the second infection with Eimeria boris.
DISCUSSION
Both drugs used appeared to be effective in suppressing m ost of the clinical signs due to E. b o v i s in the experimental calves but there were mild clinical signs in two animals treated with amprolium, and m ore severe signs such as d y s e n ter y and diarrhoea in t w o calves treated with monensin. These results agree with those of Fitzgerald and Mansfield (1973), who used monensin at the same dose rate, 1 mg kg -~ b o d y weight daily, but who treated the animals from three days before infection until 30 days after infection, and with those of McDougald (1978) who treated calves with monensin from three days before infection to 28 days after infection at two dose levels of the drug (16.5 g or 33 g per metric t on o f feed). H a m m o n d et al. (1966) r e p o r t e d t hat amprolium, given at a rate of 143 mg kg -1 b o d y weight for five days beginning on the 13th day after infection, was highly effective in controlling the disease. All the calves, with the e x c e p t i o n of one on the amprolium t reat m ent ,
176 passed numbers of fecal oocysts during the first infection. This also happened in earlier work with E. z u e r n i i infections in cattle (Stockdale and Yates, 1978} under similar experimental conditions. The same p h e n o m e n o n had been noticed in E. bovis infections in cattle by H a m m o n d et al. (1966) when they began treatment with amprolium on the 13th day after infection. McDougald (1978} reported only a " t r a c e " of oocysts passed by calves experimentally infected with E. boris and treated with monensin at a rate of 16.5 g per metric ton of feed, but he began treatment shortly before infection. The peak of oocyst production with E. bovis was not delayed in the calves of the monensin group but was delayed in calves treated with amprolium. This was noted earlier by H a m m o n d et al. (1966) in calves infected with E. boris and treated with amprolium. Slater et al. (1970) considered that this delay in the completion of the life cycle was due to a delayed development or retardation of the first generation schizont. This delayed development has also been reported to occur with E. z u e r n i i infections in calves (Stockdale and Yates, 1978). As can be seen from Fig. 1, the general character of the lines plotting weight gains for the three groups is similar. However, there was a mean loss of weight in the calves of the control group, while the mean weight of the calves of the treated groups remained steady with the calves neither gaining nor losing weight for a period of six days. In calves infected with E. z u e r n i i and treated with the same drugs using a similar regimen there was no apparent set-back in growth rates (Stockdale and Yates, 1978), however, the numbers of fecal oocysts passed were lower with consequentially less epithelial damage to the large intestine and this might account for this difference in rates of weight gain. Another plausible explanation is that both drugs were less effective against E. bovis than against E. zueruii during the treatment period and those organisms that escaped the effects of treatment caused more damage to the epithelium of the large intestine. From the results obtained here from the second infection, such as lack of clinical signs, the reduction in total fecal oocyst o u t p u t and apparent similarity of weight gains in the three groups of calves, it is concluded that the first infection with E. boris had stimulated a resistance to the challenge infection. H a m m o n d et al. (1966) reported similar protection against a challenge infection of E. bovis in calves that had been infected with the same organism in a primary infection and treated with amprolium.
ACKNOWLEDGEMENTS The authors thank Dr. D. Mitchell of ADRI(W) for his support of this work and Agriculture Canada for operating grant No. 8011. The facilities used for raising " m i n i m u m coccidiosis" calves were kindly provided by the Veterinary Infectious Diseases Organization of Saskatoon.
177 REFERENCES Fitzgerald, P.R. and Mansfield, M.E., 1973. Efficacy of monensin against bovine coccidiosis in young Holstein-Friesian calves. J. Protozool., 20: 121--126. Gibbons, J.G., 1976. Non-Pammetric Methods for Quantitative Analysis. 1st edn., Holt, Reinhart and Winston, New York. Gunther, W.C., 1964. Analysis of Variance. 1st edn., Prentice Hall, Englewood Cliffs, NJ. Hammond, D.M., Fayer, R. and Miner, M.L., 1966. Amprolium for control of experimental coccidiosis in cattle. Am. J. Vet. Res., 27: 199--206. Levine, N.D. and Ivens, V., 1970. The Coccidian Parasites (Protoxoa, Sporozoa) of Ruminants. Illinois Biological Monographs, University of Illinois Press, pp. 47 and 53. McDougald, L.R., 1978. Monensin for the prevention of coccidiosis in calves. Am. J. Vet. Res., 39: 1748--1749. Slater, R.L., Hammond, D.M. and Miner, M.L., 1970. Eimeria bovis: development in calves treated with thiamine metabolic antagonist (amprolium) in feed. Trans. Am. Microsc. Soc., 89: 55--65. Stockdale, P.H.G., 1976. Production of coccidiosis using oocysts, sporocysts and sporozoites of Eimeria zuernii, E. contorta and E. nieschulzi. Can. J. Zool., 54: 1064--1068. Stockdale, P.H.G. and Niilo, L., 1976. Production of bovine coccidiosis with E. zuernii. Can. Vet. J., 17: 35--37. Stockdale, P.H.G. and Yates, W.D.G., 1978. Resistance to Eimeria zuernii produced after chemotherapy of experimental infection in calves. Vet. Parasitol., 4: 209--214.
RESISTANCE TO EIMERIA BOVIS PRODUCED OF EXPERIMENTAL INFECTIONS IN CALVES
AFTER
171
CHEMOTHERAPY
P.H.G. STOCKDALE and ANNE SHEARD Department o f Veterinary Microbiology, University o f Saskatchewan, Saskatoon, Saskatche wan S 7N 0 WO i(Canada)
G.B. T I F F I N Animal Disease Research Institute, Box 640, Lethbridge, Alberta TIJ 3Z4 (Canada)
(Accepted for publication 13 August 1981 )
ABSTRACT
Stockdale, P.H.G., Sheard, A. and Tiffin, G.B., 1982. Resistance to Eimeria bovis produced after chemotherapy of experimental infections in calves. Vet. Parasitol., 9: 171--177. Eighteen Holstein-Friesian four week old calves were inoculated with 5 x 10 s sporocysts of Eimeria boris by stomach tube. The calves were divided into three equal groups. The first group was treated with monensin, 1 mg kg-' body weight daily, from the 10th-20th day inclusive after infection; the second group was treated with amprolium, 10 mg kg - ' body weight daily, for the same period of time and the third group acted as infected controls. Both drugs were for the most part effective in preventing clinical signs, in suppressing reduced rates of weight and in reducing oocyst production. The calves of all three groups were resistant to the second infection given 35 days after the first infection with E. bovis as measured by rates of weight gain, fecal oocyst output and lack of clinical signs. INTRODUCTION L e v i n e a n d I v e n s ( 1 9 7 0 ) d e s c r i b e d E i m e r i a z u e r n i i a n d E. b o v i s as b e i n g t h e t w o m o s t p a t h o g e n i c s p e c i e s o f c o c c i d i a in c a t t l e . I n e a r l i e r w o r k t h e e f f e c t s o f t r e a t m e n t o f E. z u e r n i i i n f e c t i o n s in c a l v e s w i t h m o n e n s i n a n d amprolium and the development of resistance to challenge infection of the calves were described (Stockdale and Yates, 1978). It was decided to do the f o l l o w i n g e x p e r i m e n t o n E. b o v i s t o see i f a s i m i l a r i n f e c t i o n a n d t r e a t m e n t r e g i m e n w i t h t h i s l a t t e r p r o t o z o a n w o u l d p r o d u c e s i m i l a r r e s u l t s t o t h o s e rep o r t e d f o r E. z u e r n i i , as a n y p r a c t i c a l p r o p h y l a c t i c t r e a t m e n t f o r c o c c i d i o s i s in c a t t l e w o u l d n e e d t o b e d i r e c t e d a g a i n s t b o t h o f t h e a b o v e o r g a n i s m s . MATERIALS AND METHODS Eighteen Holstein-Friesian calves were raised under "minimal coccidiosis" c o n d i t i o n s as d e s c r i b e d e a r l i e r ( S t o c k d a l e a n d Y a t e s , 1 9 7 8 ) . T h e c a l v e s , a p p r o x i m a t e l y f o u r w e e k s o f a g e , w e r e i n f e c t e d w i t h 5 X 1 0 s s p o r o c y s t s o f E. b o v i s 0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
172 suspended in saline via a stomach tube. All calves were given approximately 300 ml o f r u men fluid from adult cattle five days after infection, in an att e m p t to provide conventional enteric flora and fauna. This was provided because the calves had been raised under "minimal disease" conditions and had had no c o n t a c t with adult cattle, thus they had n o t acquired a " n o r m a l " intestinal biota. There was some slight risk that coccidia, ot her than the dose given, were added with the rumen fluid but this risk was taken as it was considered to be i m p o r t a n t to provide the calves with rum en organisms that would allow t h e m to make more use of the alfalfa pellets and dairy concentrate with which t hey were being fed. The calves were injected intramuscularly with 40 mg of de xa m et has one (Dexagen 5 or 2, Rogar/STB, L o n d o n , Ontario) on Days 13, 14 and 15 after infection (Stockdale and Niilo, 1976). This was used, to be consistent with the E. zuernii infections described earlier (Stockdale and Yates, 1978), as it was desirable to examine E. boris under similar conditions. Calves were infected in replicates of three animals. All were bull calves, with the ex cep tion of one free-martin heifer calf. The calves were given drugs f r o m Day 10 to Day 20 inclusive post-infection (PI), and bot h drugs were fed so that the daily dose was divided into two parts and fed in milk replacer. The first calf of each replicate was given 1.0 mg kg -1 b o d y weight of monensin (Coban, Elanco Ltd., IN), the second calf was given 10 mg kg -1 of amprolium (Amprovine, Merck, NY) and the third calf remained as an untreated, infected control. There were six calves in each t r e a t m e n t and c o n tr o l group. Calves were weighed twice weekly and from Day 13 all feces passed were collected at 24 h intervals. The total daily fecal o u t p u t of oocysts per calf was estimated as previously described (Stockdale, 1976) until Day 30 after infection. Calves were reinfected on Day 35 (PI) with 5 X 105 sporocysts of E. bovis and the various required parameters measured as in the first infection. Fecal o o c y s t o u t p u t f r om this second infection in the calves was estim a t e d from Day 13--Day 25 (49--60 days after the first infection). The calves were n o t treated with any drugs during the second infection. The oocysts of E. bovis used as a source of sporocysts t h r o u g h o u t the e x p e r i m e n t were fr om a single c o m m o n pool and, as the work was done with successive replicates, a following replicate acted as a control for oocyst infectivity for each challenge infection. RESULTS All the calves survived the experimental period but all calves in the last three replicates developed concur r ent enzootic pneumonia. As this appeared to affect all the calves, it was assumed that the comparisons made between the coccidial infections were still valid. The control calves had clinical coccidiosis during the first infection. Their feces became diarrhoeic and later contained blood, mucus, large sheets of
173 m u c o s a a n d fibrin casts. T h e r e was d e h y d r a t i o n a n d t e n e s m u s in the m o s t severely a f f e c t e d c o n t r o l calves a n d t h e s e signs o c c u r r e d at a p p r o x i m a t e l y the s a m e t i m e as p e a k o o c y s t p r o d u c t i o n . T h e clinical signs w e r e m u c h red u c e d in t h e calves t r e a t e d w i t h a m p r o l i u m . O n l y t w o o f these calves h a d mild d i a r r h o e a f r o m D a y 2 0 - - D a y 22 a f t e r i n f e c t i o n . H o w e v e r t w o calves (calves 5 and 6) o f the g r o u p t r e a t e d w i t h m o n e n s i n h a d clinical disease. T h e s e calves h a d d i a r r h o e a a n d d y s e n t e r y and t h e i r feces c o n t a i n e d casts o f epithelial cells, b u t n o n e o f t h e s e signs were as severe as t h o s e seen in t h e u n t r e a t e d c o n t r o l calves. All calves' feces w e r e o f n o r m a l c o n s i s t e n c y b y D a y 30 a f t e r i n f e c t i o n . In t h e first i n f e c t i o n , p e a k o o c y s t p r o d u c t i o n f o r E. b o v i s o c c u r r e d on D a y 21 f o r t h e calves o f t h e m o n e n s i n and c o n t r o l g r o u p s and on D a y 25 f o r t h e calves o f t h e g r o u p given a m p r o l i u m . In t h e s e c o n d i n f e c t i o n , w i t h o u t t r e a t m e n t , t h e p e a k o f o o c y s t p r o d u c t i o n was on D a y 21 for all t h r e e groups. F r o m T a b l e I it c a n be seen t h a t in t h e first i n f e c t i o n b o t h drugs d e p r e s s e d o o c y s t o u t p u t f r o m t h e calves as c o m p a r e d t o t h a t o f t h e c o n t r o l calves. T h e r e d u c t i o n in o u t p u t was statistically significantly d i f f e r e n t using t h e MannW h i t n e y U t e s t ( G i b b o n s , 1 9 7 6 ) . F r o m T a b l e II it is a p p a r e n t t h a t t h e r e was little d i f f e r e n c e in m e a n t o t a l fecal o o c y s t o u t p u t o f t h e t h r e e g r o u p s o f calves d u r i n g t h e s e c o n d i n f e c t i o n . T h e r e was n o statistically significant d i f f e r e n c e b e t w e e n t h e s e m e a n values using t h e M a n n - W h i t n e y U test. TABLE I Fecal oocyst output of calves infected with E. bovis of the first infection Replicate
1 2 3 4 5 6 Mean
Treatment Monensin
Amprolium
Control
3.961 94.11 4.22 92.42 229.42 308.04 122.032
2.05 60.15 0.00 0.11 51.46 7.53 20.222
1910.59 247.93 113.92 184.00 430.11 407.15 548.952
1 × 10 60ocysts. 2 Statistically significant (Mann-Whitney U test). T h e p e r c e n t a g e w e i g h t gains f o r the t h r e e g r o u p s o f calves f r o m D a y 1 3 D a y 30 PI f o r the first i n f e c t i o n are given in Fig. 1. T h e lines o f b e s t fit w e r e d r a w n t o t h e values b y h a n d . All t h r e e lines are similar in c h a r a c t e r , t h a t is sigmoid in shape. T h u s it a p p e a r s t h a t in t h e c o n t r o l g r o u p t h e r e was a m e a n loss in w e i g h t f r o m D a y 18 t o D a y 22 a f t e r i n f e c t i o n , w h e r e a s in t h e t w o d r u g t r e a t e d g r o u p s t h e r e was n o increase in w e i g h t b e t w e e n D a y 18 a n d D a y 24 a f t e r i n f e c t i o n . T h e d i f f e r e n c e s in w e i g h t c h a n g e b e t w e e n the t h r e e g r o u p s
174 TABLE II Fecal oocyst output of calves infected with E. bovis of the second infection Replicate
Treatment Monensin
Amprolium
Control
1 2 3 4
1.101 0.00 0.00 12.02
17.18 1.17 0.00 0.00
0.00 0.00 0.08 0.00
5
+re 2
+re
+re
6
0.00
0.00
0.00
Mean
2.103
3.063
0.023
1× 106 Oocysts. 2 Only detected on flotation. 3 Not statistically significant (Mann-Whitney U test.).
20
©
•
a _c a~
©
OControluntreatedcalves
•
:
J
Amprolium treated calves •
•
Monensin
14
16
18
20
22
treatedcalves
24
26
28
•
30
OaysAfterInfection Fig. 1. Mean weight gains of calves after the first infection with Eimeria bovis. in t h e f i r s t i n f e c t i o n w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t ( G u n t h e r , 1 9 6 4 , F t e s t ) . T h e p e r c e n t a g e w e i g h t g a i n s f o r t h e t h r e e g r o u p s o f c a l v e s in t h e s e c o n d i n f e c t i o n a r e g i v e n in F i g . 2. T h e t h r e e g r o u p s o f c a l v e s g a i n e d weight at similar rates which were not statistically significantly different (F test).
175
60
J 5O .= ~3 .c O
O
40
~
O
© Control untreated calves
O
Amprolium treated calves •
30
Monensin treated calves
14
16
18
20
22
24
26
28
•
30
Days After Infection
Fig. 2. Mean weight gains of calves after the second infection with Eimeria boris.
DISCUSSION
Both drugs used appeared to be effective in suppressing m ost of the clinical signs due to E. b o v i s in the experimental calves but there were mild clinical signs in two animals treated with amprolium, and m ore severe signs such as d y s e n ter y and diarrhoea in t w o calves treated with monensin. These results agree with those of Fitzgerald and Mansfield (1973), who used monensin at the same dose rate, 1 mg kg -~ b o d y weight daily, but who treated the animals from three days before infection until 30 days after infection, and with those of McDougald (1978) who treated calves with monensin from three days before infection to 28 days after infection at two dose levels of the drug (16.5 g or 33 g per metric t on o f feed). H a m m o n d et al. (1966) r e p o r t e d t hat amprolium, given at a rate of 143 mg kg -1 b o d y weight for five days beginning on the 13th day after infection, was highly effective in controlling the disease. All the calves, with the e x c e p t i o n of one on the amprolium t reat m ent ,
176 passed numbers of fecal oocysts during the first infection. This also happened in earlier work with E. z u e r n i i infections in cattle (Stockdale and Yates, 1978} under similar experimental conditions. The same p h e n o m e n o n had been noticed in E. bovis infections in cattle by H a m m o n d et al. (1966) when they began treatment with amprolium on the 13th day after infection. McDougald (1978} reported only a " t r a c e " of oocysts passed by calves experimentally infected with E. boris and treated with monensin at a rate of 16.5 g per metric ton of feed, but he began treatment shortly before infection. The peak of oocyst production with E. bovis was not delayed in the calves of the monensin group but was delayed in calves treated with amprolium. This was noted earlier by H a m m o n d et al. (1966) in calves infected with E. boris and treated with amprolium. Slater et al. (1970) considered that this delay in the completion of the life cycle was due to a delayed development or retardation of the first generation schizont. This delayed development has also been reported to occur with E. z u e r n i i infections in calves (Stockdale and Yates, 1978). As can be seen from Fig. 1, the general character of the lines plotting weight gains for the three groups is similar. However, there was a mean loss of weight in the calves of the control group, while the mean weight of the calves of the treated groups remained steady with the calves neither gaining nor losing weight for a period of six days. In calves infected with E. z u e r n i i and treated with the same drugs using a similar regimen there was no apparent set-back in growth rates (Stockdale and Yates, 1978), however, the numbers of fecal oocysts passed were lower with consequentially less epithelial damage to the large intestine and this might account for this difference in rates of weight gain. Another plausible explanation is that both drugs were less effective against E. bovis than against E. zueruii during the treatment period and those organisms that escaped the effects of treatment caused more damage to the epithelium of the large intestine. From the results obtained here from the second infection, such as lack of clinical signs, the reduction in total fecal oocyst o u t p u t and apparent similarity of weight gains in the three groups of calves, it is concluded that the first infection with E. boris had stimulated a resistance to the challenge infection. H a m m o n d et al. (1966) reported similar protection against a challenge infection of E. bovis in calves that had been infected with the same organism in a primary infection and treated with amprolium.
ACKNOWLEDGEMENTS The authors thank Dr. D. Mitchell of ADRI(W) for his support of this work and Agriculture Canada for operating grant No. 8011. The facilities used for raising " m i n i m u m coccidiosis" calves were kindly provided by the Veterinary Infectious Diseases Organization of Saskatoon.
177 REFERENCES Fitzgerald, P.R. and Mansfield, M.E., 1973. Efficacy of monensin against bovine coccidiosis in young Holstein-Friesian calves. J. Protozool., 20: 121--126. Gibbons, J.G., 1976. Non-Pammetric Methods for Quantitative Analysis. 1st edn., Holt, Reinhart and Winston, New York. Gunther, W.C., 1964. Analysis of Variance. 1st edn., Prentice Hall, Englewood Cliffs, NJ. Hammond, D.M., Fayer, R. and Miner, M.L., 1966. Amprolium for control of experimental coccidiosis in cattle. Am. J. Vet. Res., 27: 199--206. Levine, N.D. and Ivens, V., 1970. The Coccidian Parasites (Protoxoa, Sporozoa) of Ruminants. Illinois Biological Monographs, University of Illinois Press, pp. 47 and 53. McDougald, L.R., 1978. Monensin for the prevention of coccidiosis in calves. Am. J. Vet. Res., 39: 1748--1749. Slater, R.L., Hammond, D.M. and Miner, M.L., 1970. Eimeria bovis: development in calves treated with thiamine metabolic antagonist (amprolium) in feed. Trans. Am. Microsc. Soc., 89: 55--65. Stockdale, P.H.G., 1976. Production of coccidiosis using oocysts, sporocysts and sporozoites of Eimeria zuernii, E. contorta and E. nieschulzi. Can. J. Zool., 54: 1064--1068. Stockdale, P.H.G. and Niilo, L., 1976. Production of bovine coccidiosis with E. zuernii. Can. Vet. J., 17: 35--37. Stockdale, P.H.G. and Yates, W.D.G., 1978. Resistance to Eimeria zuernii produced after chemotherapy of experimental infection in calves. Vet. Parasitol., 4: 209--214.