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Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken
Veterinary Parasitology, 37 ( 1 9 9 0 ) 9 3 - 1 0 0 Elsevier Science Publishers B.V., A m s t e r d a m
93
Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken V. G u y o n n e t , J.K. Johnson and P.L. Long Department of Poultry Science, University of Georgia, Athens, GA 30602 (U.S.A.) (Accepted for publication 12 April 1990 )
ABSTRACT Guyonnet, V., Johnson, J.K. and Long, P.L., 1990. Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken. Vet. ParasitoL, 37: 93-100. Broiler chickens in battery pens were either fed a diet containing 100 ppm lasalocid or no drug for 24 h prior to inoculation with sporulated oocysts of Eimeria tenella or Eimeria acervulina. Different groups of birds remained on medicated feed for 24, 48, 72, 96, 120 or 144 h after inoculation. Conversely, other groups started on an unmedicated diet, were given medicated feed at different times after oocyst inoculation. Starting lasalocid medication 24 h (E. tenella) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain. There was no significant difference in performance of birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. Starting lasalocid medication at 96 or 120 h did not suppress but rather reduced oocyst production.
INTRODUCTION
Lasalocid, a polyether monocarboxylic acid ionophorous antibiotic, was first reported to possess anticoccidial activity against species of Eimeria in broiler chickens by Mitrovic and Schildknecht (1973, 1974). The anticoccidial activity was further confirmed by other workers (Reid et al., 1975; Frigg and Schramm, 1977 ). Although the properties of the ionophores have been known for some time (Pressman, 1973, 1976 ), the knowledge of the mode of action and the stages affected by lasalocid is incomplete. Smith et al. (1981 ) reported that pretreatment ofEimeria tenella sporozoites with lasalocid inhibited invasion and subsequent development in chick kidney cell cultures. However, the viability of lasalocid-treated extracellular sporozoites has not been tested in vivo. Recently, Ball et al. (1990) studied the effect of lasalocid on the development of E. tenella in chicken embryos. These workers found that pretreatment of sporozoites with concentrations of lasalocid ranging from 0.1 to 10/~g m l - ~ reduced sporozoite viability as judged by oocyst production. They also observed that injection of lasalocid 92-93 h after inoculation reduced the oocyst production, indicating activity against the later stages of 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
94
V. GUYONNET ET AL.
the life cycle. We have used the technique of delayed medication in a similar way to that of Long and Jeffers (1982) to study the stage of action oflasalocid against E. tenella and Eimeria acervulina in the chicken. MATERIALS AND METHODS
Peterson × Arbor Acre broiler chickens were reared under conditions aimed to keep them coccidia-free to 2 weeks of age. For each trial, the birds were wingbanded and randomly allocated by weight to each treatment group (four replicates of five or seven birds). The treatment groups were as follows: unmedicated uninfected, unmedicated infected, withdrawal of the drug at 0, 24, 48, 72, 96, 120 and 144 h after inoculation and addition of the drug at 0, 24, 48, 72, 96, 120 and 144 h after inoculation. The birds were transferred to battery pens 24 h before inoculation, allowing some birds to receive medicated feed prior to the coccidia exposure. Standard UGA broiler mash ration was added with 100 ppm lasalocid (AvatecTM,Hoffmann-La Roche). Birds were inoculated at 0 h via the crop with E. tenella ( 105 sporulated oocysts) or E. acervulina ( 1.5 X 106 sporulated oocysts). At 24 h intervals, medicated diets were withdrawn and replaced by unmedicated ones. Likewise, groups initially given an unmedicated diet were started on a medicated diet at different times after inoculation. At Day 6 post inoculation (PI), weight gains and feed conversion ratios were calculated. Lesions scores (Johnson and Reid, 1970) were recorded on all the birds in the E. acervulina experiment whereas for the trial with E. tenella, lesion scores were recorded only on four birds per pen or 16 birds per treatment. The remaining birds were used for the cecal oocyst count on Day 7 PI. Fecal oocyst output on Day 6 PI was also recorded for the E. acervulina experiment according to Long et al. ( 1976 ). Data were analyzed using the general linear model (Statistical Analysis System, 1982 ) and means were analyzed using Duncan's multiple range test using a significance base of P < 0.05. RESULTS
Eirneria tenella experiment The performance of the birds infected with 105 sporulated oocysts is given in Table 1. There was no significant difference in the weight gain between uninfected controls and groups withdrawn from medication at 24 h or later. When medication was interrupted at the time of inoculation, the weight gain was significantly reduced and the weight gain was not significantly different from that of the infected controls. There was no significant difference for the feed conversion ratio between all medicated groups except for the one with-
ACTION OF LASALOCIDON EIMERIA
95
TABLE 1
Performance of birds infected with Eimeria tenella and treated with lasalocid ( 100 p p m ) at different times in relation to inoculation with oocysts Treatment
Weight gain ( g )
L e s i o n score ~
F e e d conversion
0 3.3
1.55 d~ 1.88 a
184.9 abed
0.4
1.56 ode
24 h 209.3 ab 48 h 191.2 abcd 72 h 1 7 8 . 0 bcd 96 h 160.0 de 120 h 162.1 de 144 h 146.1 e T i m e o f withdrawal of medication 4 0 h 169.2 ode 24 h 194.8 ab~ 48 h 202.8 ab 72 h 195.1 abc 96 h 200.1 abe 120 h 210.3 ab 144 h 189.1 abed
0.5 1.8 1.7 2.8 3.0 3.3
1.54 dc 1.61 bcd~
2.7 1.4 0.3 0.7 0.3 O. 1 0
1.69 bed 1.57 ode 1.46 e 1.51 e 1.53 e 1.49 ~ 1.52 ~
Controls, unmedicated Uninfected 211.72a Infected 146.1 e Time of initiating medication 3 0 h
1 . 5 9 cae
1.75 ab 1.72 bc 1.88 a
I M a x i m u m score equals 4.0. 2Values w i t h i n a column with a different superscript are different at P < 0.05.
3Medication was continuous once started. 4Medication started 24 h before inoculation of oocysts.
drawn at 0 h. Unmedicated infected birds showed the highest lesion score and withdrawal of the drug at 24-h intervals gradually reduced the scores. The cecal oocyst count when the drug was withdrawn at 0 h was similar to that of the unmedicated controls (Fig. 1 ). Cecal oocyst counts in birds withdrawn from medication at 24 or 48 h PI were about 50% lower than those of the infected controls. For groups withdrawn at 72, 96, 120 and 144 h, the counts were reduced by 91, 96, 100 and 96%, respectively. No significant differences in weight gains were seen in the groups given the drug through 48 h when compared with the uninfected controls (Table 1 ). However, addition of the drug after 48 h did result in significantly lower weight gains. The feed conversion ratio was not significantly different between the uninfected controls and the groups given the drug at 0, 24, 48 and 72 h. When given later, the drug did not significantly improve the feed conversion ratio when compared with the infected controls (Table 1 ). Starting lasalocid treatment at 24-h intervals after oocyst inoculation gradually increased the intensity of the lesions seen on Day 6 PI. When medication was started up to 24 h after inoculation, the oocyst
96
V. G U Y O N N E T E T AL.
1O0 T J / 80
r ~ l E. acervulina ~ E. tenella
60
~
20
o
o
,I
-24
0
[7, 2'4
48
7'2
9'6
120
44
Time of withdrawal (Hrs)
Fig. 1. Oocyst output reduction (%) after withdrawing lasalocid medication at different times after oocyst inoculation. 100-
[2225] E. E.
aeervulina tenella
80
~? 40 o 0
'
'
:
'
'
-24
6
24
48
7'2
9'6
I
20
144
Time of addition (Hrs)
Fig. 2. Oocyst output reduction (%) in groups of chickens given lasalocid continuously starting at different times after oocyst inoculation. production was reduced by 84 and 91% (Fig. 2 ). W h e n medication was started at 48 or 72 h after infection, the drug still had a m a r k e d effect on the oocyst production (reduction of 63% and 72% respectively). Starting medication 72 h after infection also reduced the oocyst production.
Eimeria acervulina experiment The withdrawal of lasalocid after 72 h did not significantly decrease the weight gain when c o m p a r e d with the uninfected controls (Table 2 ). On the other hand, birds withdrawn from medication at the time of exposure to coccidia expressed a weight gain not significantly different from that of the in-
97
ACTION OF LASALOCID ON EIMERIA
TABLE2
Performance of birds infected w i t h Eimeria acervulina and treated with lasalocid ( 100 p p m ) at different times in relation to inoculation with oocysts Treatment Controls, unmedicated Uninfected
Weight gain ( g )
252.92a Infected 128.5 ~ T i m e o f initiating medication 3 0 h 246.7 a 24 h 242.1 a 48 h 205.4 b 72 h 152.7 de 96 h 133.4 d~ 120 h 158.5 cd 144 h 128.5 ~ T i m e o f withdrawal of medication 4 0 h 1 5 3 . 0 de 24 h 184.5 bc 48 h 190.6 b 72 h 237.6 a 96 h 238.4 a 120 h 235.1 a 144 h 247.2 a
Lesion score ~
Feed conversion
0 3.8
1.50 ~ 2.34 ~
0.2 0.9 0.9 3.5 3.1 3.4 3.8
1.53 g 1.52 ~ 1.84 cd 2.02 bc 2.15 ~b 1.94 bed 2.34 ~
3.5 3.0 2.4 0.8 0.2 0 0.1
2. l 0 b 1.80 cde 1.77 def 1.56 fg 1.61 ~fg 1.55 fg 1.52 g
~ M a x i m u m score equals 4.0. 2Values within a column with a different superscript are different at P < 0.05. 3Medication was continuous once started. 4Medication started 24 h before inoculation of oocysts.
fected controls. The weight gain was significantly different between groups taken off medication at 24-48 h and both control groups. Similarly, when the medicated feed was withdrawn after 72 h, there was no significant difference in the feed conversion ratio between the medicated groups and the uninfected controls. All medicated groups had a significantly better feed conversion ratio when compared with that of the infected controls. The lesions were mild or absent when the drug was withdrawn after 72 h. When withdrawn during the first 24 h, the drug did not prevent the presence of severe lesions on Day 6 PI. Withdrawal oflasalocid up to 48 h after inoculation gave rise to oocyst counts similar to those of the infected controls (Fig. 1 ). The oocyst production was reduced by 90% and 97% when the drug was withdrawn at 72 h and 96 h, respectively. No oocysts were detected when the birds were kept on medication for 120 or 144 h. When the drug was given up to 24 h after inoculation, the weight gain was not significantly different between these two groups and the uninfected controis (Table 2 ). The weight gain observed for birds given the drug at 48 and
98
V. GUYONNET ET AL.
120 h appeared significantly different from those of both control groups. For the other treatment groups, the gain at Day 6 PI was not significantly different from that of infected controls. When lasalocid was added at 0 or 24 h PI, the feed conversion ratio was not significantly different between these two groups and the uninfected controls. For the other treatment groups, the feed conversion ratio was somewhat intermediate between those of the control groups. Starting medication up to 48 h PI reduced the lesions observed on Day 6 PI. Starting lasalocid treatment up to 48 h after inoculation reduced the oocyst production by more than 90% (Fig. 2). Initiating drug treatment at 72 and 96 h had some effect in reducing the oocyst production (79 and 76% reduction, respectively). Giving the drug as late as 120 h also reduced oocyst production ( 59% reduction). DISCUSSION
Since the discovery of valinomycin, the first ionophore recognized as such, it has been found that ionophores catalyze transport by enveloping an ion and diffusing across the membrane as a cation complex (Pressman, 1973 ). This ability to carry ions across membranes is also considered to be the mechanism of action of anticoccidial ionophorous antibiotics (McDougald, 1982 ). Most of the studies on the mode and site of action of the ionophores have been made with monensin and results are often extrapolated to more recently discovered ionophores. In our experiments, we chose lasalocid, an ionophore commonly used in the field, because little is known of its stage of action in vivo. The use of delayed medication appeared to be an appropriate means of studying in vivo the stages of action of the drug, as previously mentioned by Long and Jeffers (1982). Starting medication with lasalocid 100 p p m up to 24 h (E. tenella ) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain of the birds at Day 6 PI. Likewise, there was no significant difference in the performance of the birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. These results indicated that lasalocid is active against early stages of parasite development (sporozoites and merozoites). Our findings supported previous studies showing lasalocid active on sporozoites (Smith and Strout, 1979; Smith et al., 1981; Long and Jeffers, 1982) and merozoites (Mehlhorn et al., 1983 ). They were also in accord with a recent demonstration by Ball et al. (1990) that concentrations of 0.1, 1.0 and 10.0 #g ml-1 of lasalocid reduced sporozoite viability as judged by the oocyst production in ovo. By combining the results obtained by addition or withdrawal of lasalocid, it appeared that lasalocid activity was optimal between 24 and 48 h PI (E. tenella) and 48 and 72 h (E. acervulina ). The difference in activity between the two species is not fully understood although a similar finding was previously reported by Long and Jeffers (1982) working with monensin. They noted during a delayed-
ACTION OF LASALOCID ON EIMERIA
99
medication type experiment that monensin added at 24 h PI prevented the appearance of lesions with E. necatrix but not with E. tenella. In considering the activity of the drug against oocyst production, it appears that starting medication with lasalocid at 96 and 120 h reduced the output (Fig. 2 ). These results suggest that lasalocid may also act on later stages of development (e.g. meronts, merozoites and perhaps gamonts). Long and Jeffers (1982) found similar results with monensin and concluded that low numbers of oocysts probably reflected the direct action of the drug on gametogony mediated through an effect on late generations of merozoites. Like monensin, lasalocid appeared to act on merozoites of late meront generations. At both 96 and 120 h PI, the reduction in oocyst output was higher for E. acervulina than for E. tenella. Similar differences between species were also observed in studies on monensin (Long and Jeffers, 1982). Recently, Ball et al. (1990) also found that lasalocid treatment of chicken embryos 92-93 h after inoculation with E. tenella reduced the oocyst production by up to 97% when 500/tg per embryo were given. The present results support previous work and showed that lasalocid was active on both early and late stages of the parasite. Such characteristics enhance its potency for prophylactic control of coccidiosis. Because of the epidemiological significance of reduction of oocyst output (Long and Jeffers, 1982 ), our results suggest also that lasalocid may be used in combination with other drugs for the treatment of coccidiosis. Further studies are needed to compare the ability of various ionophores to reduce oocyst output when given late in the life cycle.
REFERENCES Ball, S.J., Pittilo, R.M., Johnson, J. and Long, P.L., 1990. Effect of lasalocid on the development ofEimeria tenella in chicken embryos. Vet. Parasitol., 36: 337-341. Frigg, M. and Schramm, H., 1977. Comparative anticoccidial activity of lasalocid sodium (Avatec R) in chicks: Efficacy against European strains of coccidia. Arch. Gefluegelkd., 41: 31-34. Johnson, J. and Reid, W.M., 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments. Exp. Parasitol., 28: 30-36. Long, P.L. and Jeffers, T.K., 1982. Studies on the stage of action of ionophorous antibiotics against Eimeria. J. Parasitol., 68:363-371. Long, P.L., Joyner, L.P., Millard, B.J. and Norton, C.C., 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat., 6:201-217. McDougald, L.R., 1982. Chemotherapy of coccidiosis. In: P.L. Long (Editor), Biology of the Coccidia. University Park Press, Baltimore, MD, pp. 373-428. Mehlhorn, H., Pooch, H. and Raether, W., 1983. The action of polyether ionophorous antibiotics (monensin, salinomycin, lasalocid) on developmental stages ofEimeria tenella (Coccidia, Sporozoa) in vivo and in vitro: Study by light and electron microscopy. Z. Parasitenkd., 69:457-471.
1O0
v. GUYONNETET AL.
Mitrovic, M. and Schildknecht, E.G., 1973. Anticoccidial activity of antibiotic X-537A in chickens. Poultry Sci., 52: 2065. Mitrovic, M. and Schildknecht, E.G., 1974. Anticoccidial activity of lasalocid (X-537A) in chicks. Poultry Sci., 53: 1448-1455. Pressman, B.C., 1973. Properties of ionophores with broad range cation selectivity. Fed. Proc., 32: 1698-1703. Pressman, B.C., 1976. Biological applications of ionophores. Annu. Rev. Biochem., 45: 501530. Reid, W.M., Johnson, J. and Dick, J., 1975. Anticoccidial activity of lasalocid in control of moderate and severe coccidiosis. Avian Dis., 19:12-18. Statistical Analysis System, 1982. User's guide. SAS Institute, Inc., Cary, NY. Smith, C.K. II and Strout, R.G., 1979. Eimeria tenella: Accumulation and retention of anticoccidial ionophores by extracellular sporozoites. Exp. Parasitol., 48: 325-330. Smith, C.K. II, Galloway, R.B. and White, S.L., 1981. Effect of ionophores on survival, penetration and development ofEimeria tenella sporozoites in vitro. J. Parasitol., 67:511-516.
93
Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken V. G u y o n n e t , J.K. Johnson and P.L. Long Department of Poultry Science, University of Georgia, Athens, GA 30602 (U.S.A.) (Accepted for publication 12 April 1990 )
ABSTRACT Guyonnet, V., Johnson, J.K. and Long, P.L., 1990. Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken. Vet. ParasitoL, 37: 93-100. Broiler chickens in battery pens were either fed a diet containing 100 ppm lasalocid or no drug for 24 h prior to inoculation with sporulated oocysts of Eimeria tenella or Eimeria acervulina. Different groups of birds remained on medicated feed for 24, 48, 72, 96, 120 or 144 h after inoculation. Conversely, other groups started on an unmedicated diet, were given medicated feed at different times after oocyst inoculation. Starting lasalocid medication 24 h (E. tenella) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain. There was no significant difference in performance of birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. Starting lasalocid medication at 96 or 120 h did not suppress but rather reduced oocyst production.
INTRODUCTION
Lasalocid, a polyether monocarboxylic acid ionophorous antibiotic, was first reported to possess anticoccidial activity against species of Eimeria in broiler chickens by Mitrovic and Schildknecht (1973, 1974). The anticoccidial activity was further confirmed by other workers (Reid et al., 1975; Frigg and Schramm, 1977 ). Although the properties of the ionophores have been known for some time (Pressman, 1973, 1976 ), the knowledge of the mode of action and the stages affected by lasalocid is incomplete. Smith et al. (1981 ) reported that pretreatment ofEimeria tenella sporozoites with lasalocid inhibited invasion and subsequent development in chick kidney cell cultures. However, the viability of lasalocid-treated extracellular sporozoites has not been tested in vivo. Recently, Ball et al. (1990) studied the effect of lasalocid on the development of E. tenella in chicken embryos. These workers found that pretreatment of sporozoites with concentrations of lasalocid ranging from 0.1 to 10/~g m l - ~ reduced sporozoite viability as judged by oocyst production. They also observed that injection of lasalocid 92-93 h after inoculation reduced the oocyst production, indicating activity against the later stages of 0304-4017/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
94
V. GUYONNET ET AL.
the life cycle. We have used the technique of delayed medication in a similar way to that of Long and Jeffers (1982) to study the stage of action oflasalocid against E. tenella and Eimeria acervulina in the chicken. MATERIALS AND METHODS
Peterson × Arbor Acre broiler chickens were reared under conditions aimed to keep them coccidia-free to 2 weeks of age. For each trial, the birds were wingbanded and randomly allocated by weight to each treatment group (four replicates of five or seven birds). The treatment groups were as follows: unmedicated uninfected, unmedicated infected, withdrawal of the drug at 0, 24, 48, 72, 96, 120 and 144 h after inoculation and addition of the drug at 0, 24, 48, 72, 96, 120 and 144 h after inoculation. The birds were transferred to battery pens 24 h before inoculation, allowing some birds to receive medicated feed prior to the coccidia exposure. Standard UGA broiler mash ration was added with 100 ppm lasalocid (AvatecTM,Hoffmann-La Roche). Birds were inoculated at 0 h via the crop with E. tenella ( 105 sporulated oocysts) or E. acervulina ( 1.5 X 106 sporulated oocysts). At 24 h intervals, medicated diets were withdrawn and replaced by unmedicated ones. Likewise, groups initially given an unmedicated diet were started on a medicated diet at different times after inoculation. At Day 6 post inoculation (PI), weight gains and feed conversion ratios were calculated. Lesions scores (Johnson and Reid, 1970) were recorded on all the birds in the E. acervulina experiment whereas for the trial with E. tenella, lesion scores were recorded only on four birds per pen or 16 birds per treatment. The remaining birds were used for the cecal oocyst count on Day 7 PI. Fecal oocyst output on Day 6 PI was also recorded for the E. acervulina experiment according to Long et al. ( 1976 ). Data were analyzed using the general linear model (Statistical Analysis System, 1982 ) and means were analyzed using Duncan's multiple range test using a significance base of P < 0.05. RESULTS
Eirneria tenella experiment The performance of the birds infected with 105 sporulated oocysts is given in Table 1. There was no significant difference in the weight gain between uninfected controls and groups withdrawn from medication at 24 h or later. When medication was interrupted at the time of inoculation, the weight gain was significantly reduced and the weight gain was not significantly different from that of the infected controls. There was no significant difference for the feed conversion ratio between all medicated groups except for the one with-
ACTION OF LASALOCIDON EIMERIA
95
TABLE 1
Performance of birds infected with Eimeria tenella and treated with lasalocid ( 100 p p m ) at different times in relation to inoculation with oocysts Treatment
Weight gain ( g )
L e s i o n score ~
F e e d conversion
0 3.3
1.55 d~ 1.88 a
184.9 abed
0.4
1.56 ode
24 h 209.3 ab 48 h 191.2 abcd 72 h 1 7 8 . 0 bcd 96 h 160.0 de 120 h 162.1 de 144 h 146.1 e T i m e o f withdrawal of medication 4 0 h 169.2 ode 24 h 194.8 ab~ 48 h 202.8 ab 72 h 195.1 abc 96 h 200.1 abe 120 h 210.3 ab 144 h 189.1 abed
0.5 1.8 1.7 2.8 3.0 3.3
1.54 dc 1.61 bcd~
2.7 1.4 0.3 0.7 0.3 O. 1 0
1.69 bed 1.57 ode 1.46 e 1.51 e 1.53 e 1.49 ~ 1.52 ~
Controls, unmedicated Uninfected 211.72a Infected 146.1 e Time of initiating medication 3 0 h
1 . 5 9 cae
1.75 ab 1.72 bc 1.88 a
I M a x i m u m score equals 4.0. 2Values w i t h i n a column with a different superscript are different at P < 0.05.
3Medication was continuous once started. 4Medication started 24 h before inoculation of oocysts.
drawn at 0 h. Unmedicated infected birds showed the highest lesion score and withdrawal of the drug at 24-h intervals gradually reduced the scores. The cecal oocyst count when the drug was withdrawn at 0 h was similar to that of the unmedicated controls (Fig. 1 ). Cecal oocyst counts in birds withdrawn from medication at 24 or 48 h PI were about 50% lower than those of the infected controls. For groups withdrawn at 72, 96, 120 and 144 h, the counts were reduced by 91, 96, 100 and 96%, respectively. No significant differences in weight gains were seen in the groups given the drug through 48 h when compared with the uninfected controls (Table 1 ). However, addition of the drug after 48 h did result in significantly lower weight gains. The feed conversion ratio was not significantly different between the uninfected controls and the groups given the drug at 0, 24, 48 and 72 h. When given later, the drug did not significantly improve the feed conversion ratio when compared with the infected controls (Table 1 ). Starting lasalocid treatment at 24-h intervals after oocyst inoculation gradually increased the intensity of the lesions seen on Day 6 PI. When medication was started up to 24 h after inoculation, the oocyst
96
V. G U Y O N N E T E T AL.
1O0 T J / 80
r ~ l E. acervulina ~ E. tenella
60
~
20
o
o
,I
-24
0
[7, 2'4
48
7'2
9'6
120
44
Time of withdrawal (Hrs)
Fig. 1. Oocyst output reduction (%) after withdrawing lasalocid medication at different times after oocyst inoculation. 100-
[2225] E. E.
aeervulina tenella
80
~? 40 o 0
'
'
:
'
'
-24
6
24
48
7'2
9'6
I
20
144
Time of addition (Hrs)
Fig. 2. Oocyst output reduction (%) in groups of chickens given lasalocid continuously starting at different times after oocyst inoculation. production was reduced by 84 and 91% (Fig. 2 ). W h e n medication was started at 48 or 72 h after infection, the drug still had a m a r k e d effect on the oocyst production (reduction of 63% and 72% respectively). Starting medication 72 h after infection also reduced the oocyst production.
Eimeria acervulina experiment The withdrawal of lasalocid after 72 h did not significantly decrease the weight gain when c o m p a r e d with the uninfected controls (Table 2 ). On the other hand, birds withdrawn from medication at the time of exposure to coccidia expressed a weight gain not significantly different from that of the in-
97
ACTION OF LASALOCID ON EIMERIA
TABLE2
Performance of birds infected w i t h Eimeria acervulina and treated with lasalocid ( 100 p p m ) at different times in relation to inoculation with oocysts Treatment Controls, unmedicated Uninfected
Weight gain ( g )
252.92a Infected 128.5 ~ T i m e o f initiating medication 3 0 h 246.7 a 24 h 242.1 a 48 h 205.4 b 72 h 152.7 de 96 h 133.4 d~ 120 h 158.5 cd 144 h 128.5 ~ T i m e o f withdrawal of medication 4 0 h 1 5 3 . 0 de 24 h 184.5 bc 48 h 190.6 b 72 h 237.6 a 96 h 238.4 a 120 h 235.1 a 144 h 247.2 a
Lesion score ~
Feed conversion
0 3.8
1.50 ~ 2.34 ~
0.2 0.9 0.9 3.5 3.1 3.4 3.8
1.53 g 1.52 ~ 1.84 cd 2.02 bc 2.15 ~b 1.94 bed 2.34 ~
3.5 3.0 2.4 0.8 0.2 0 0.1
2. l 0 b 1.80 cde 1.77 def 1.56 fg 1.61 ~fg 1.55 fg 1.52 g
~ M a x i m u m score equals 4.0. 2Values within a column with a different superscript are different at P < 0.05. 3Medication was continuous once started. 4Medication started 24 h before inoculation of oocysts.
fected controls. The weight gain was significantly different between groups taken off medication at 24-48 h and both control groups. Similarly, when the medicated feed was withdrawn after 72 h, there was no significant difference in the feed conversion ratio between the medicated groups and the uninfected controls. All medicated groups had a significantly better feed conversion ratio when compared with that of the infected controls. The lesions were mild or absent when the drug was withdrawn after 72 h. When withdrawn during the first 24 h, the drug did not prevent the presence of severe lesions on Day 6 PI. Withdrawal oflasalocid up to 48 h after inoculation gave rise to oocyst counts similar to those of the infected controls (Fig. 1 ). The oocyst production was reduced by 90% and 97% when the drug was withdrawn at 72 h and 96 h, respectively. No oocysts were detected when the birds were kept on medication for 120 or 144 h. When the drug was given up to 24 h after inoculation, the weight gain was not significantly different between these two groups and the uninfected controis (Table 2 ). The weight gain observed for birds given the drug at 48 and
98
V. GUYONNET ET AL.
120 h appeared significantly different from those of both control groups. For the other treatment groups, the gain at Day 6 PI was not significantly different from that of infected controls. When lasalocid was added at 0 or 24 h PI, the feed conversion ratio was not significantly different between these two groups and the uninfected controls. For the other treatment groups, the feed conversion ratio was somewhat intermediate between those of the control groups. Starting medication up to 48 h PI reduced the lesions observed on Day 6 PI. Starting lasalocid treatment up to 48 h after inoculation reduced the oocyst production by more than 90% (Fig. 2). Initiating drug treatment at 72 and 96 h had some effect in reducing the oocyst production (79 and 76% reduction, respectively). Giving the drug as late as 120 h also reduced oocyst production ( 59% reduction). DISCUSSION
Since the discovery of valinomycin, the first ionophore recognized as such, it has been found that ionophores catalyze transport by enveloping an ion and diffusing across the membrane as a cation complex (Pressman, 1973 ). This ability to carry ions across membranes is also considered to be the mechanism of action of anticoccidial ionophorous antibiotics (McDougald, 1982 ). Most of the studies on the mode and site of action of the ionophores have been made with monensin and results are often extrapolated to more recently discovered ionophores. In our experiments, we chose lasalocid, an ionophore commonly used in the field, because little is known of its stage of action in vivo. The use of delayed medication appeared to be an appropriate means of studying in vivo the stages of action of the drug, as previously mentioned by Long and Jeffers (1982). Starting medication with lasalocid 100 p p m up to 24 h (E. tenella ) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain of the birds at Day 6 PI. Likewise, there was no significant difference in the performance of the birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. These results indicated that lasalocid is active against early stages of parasite development (sporozoites and merozoites). Our findings supported previous studies showing lasalocid active on sporozoites (Smith and Strout, 1979; Smith et al., 1981; Long and Jeffers, 1982) and merozoites (Mehlhorn et al., 1983 ). They were also in accord with a recent demonstration by Ball et al. (1990) that concentrations of 0.1, 1.0 and 10.0 #g ml-1 of lasalocid reduced sporozoite viability as judged by the oocyst production in ovo. By combining the results obtained by addition or withdrawal of lasalocid, it appeared that lasalocid activity was optimal between 24 and 48 h PI (E. tenella) and 48 and 72 h (E. acervulina ). The difference in activity between the two species is not fully understood although a similar finding was previously reported by Long and Jeffers (1982) working with monensin. They noted during a delayed-
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medication type experiment that monensin added at 24 h PI prevented the appearance of lesions with E. necatrix but not with E. tenella. In considering the activity of the drug against oocyst production, it appears that starting medication with lasalocid at 96 and 120 h reduced the output (Fig. 2 ). These results suggest that lasalocid may also act on later stages of development (e.g. meronts, merozoites and perhaps gamonts). Long and Jeffers (1982) found similar results with monensin and concluded that low numbers of oocysts probably reflected the direct action of the drug on gametogony mediated through an effect on late generations of merozoites. Like monensin, lasalocid appeared to act on merozoites of late meront generations. At both 96 and 120 h PI, the reduction in oocyst output was higher for E. acervulina than for E. tenella. Similar differences between species were also observed in studies on monensin (Long and Jeffers, 1982). Recently, Ball et al. (1990) also found that lasalocid treatment of chicken embryos 92-93 h after inoculation with E. tenella reduced the oocyst production by up to 97% when 500/tg per embryo were given. The present results support previous work and showed that lasalocid was active on both early and late stages of the parasite. Such characteristics enhance its potency for prophylactic control of coccidiosis. Because of the epidemiological significance of reduction of oocyst output (Long and Jeffers, 1982 ), our results suggest also that lasalocid may be used in combination with other drugs for the treatment of coccidiosis. Further studies are needed to compare the ability of various ionophores to reduce oocyst output when given late in the life cycle.
REFERENCES Ball, S.J., Pittilo, R.M., Johnson, J. and Long, P.L., 1990. Effect of lasalocid on the development ofEimeria tenella in chicken embryos. Vet. Parasitol., 36: 337-341. Frigg, M. and Schramm, H., 1977. Comparative anticoccidial activity of lasalocid sodium (Avatec R) in chicks: Efficacy against European strains of coccidia. Arch. Gefluegelkd., 41: 31-34. Johnson, J. and Reid, W.M., 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments. Exp. Parasitol., 28: 30-36. Long, P.L. and Jeffers, T.K., 1982. Studies on the stage of action of ionophorous antibiotics against Eimeria. J. Parasitol., 68:363-371. Long, P.L., Joyner, L.P., Millard, B.J. and Norton, C.C., 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat., 6:201-217. McDougald, L.R., 1982. Chemotherapy of coccidiosis. In: P.L. Long (Editor), Biology of the Coccidia. University Park Press, Baltimore, MD, pp. 373-428. Mehlhorn, H., Pooch, H. and Raether, W., 1983. The action of polyether ionophorous antibiotics (monensin, salinomycin, lasalocid) on developmental stages ofEimeria tenella (Coccidia, Sporozoa) in vivo and in vitro: Study by light and electron microscopy. Z. Parasitenkd., 69:457-471.
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Mitrovic, M. and Schildknecht, E.G., 1973. Anticoccidial activity of antibiotic X-537A in chickens. Poultry Sci., 52: 2065. Mitrovic, M. and Schildknecht, E.G., 1974. Anticoccidial activity of lasalocid (X-537A) in chicks. Poultry Sci., 53: 1448-1455. Pressman, B.C., 1973. Properties of ionophores with broad range cation selectivity. Fed. Proc., 32: 1698-1703. Pressman, B.C., 1976. Biological applications of ionophores. Annu. Rev. Biochem., 45: 501530. Reid, W.M., Johnson, J. and Dick, J., 1975. Anticoccidial activity of lasalocid in control of moderate and severe coccidiosis. Avian Dis., 19:12-18. Statistical Analysis System, 1982. User's guide. SAS Institute, Inc., Cary, NY. Smith, C.K. II and Strout, R.G., 1979. Eimeria tenella: Accumulation and retention of anticoccidial ionophores by extracellular sporozoites. Exp. Parasitol., 48: 325-330. Smith, C.K. II, Galloway, R.B. and White, S.L., 1981. Effect of ionophores on survival, penetration and development ofEimeria tenella sporozoites in vitro. J. Parasitol., 67:511-516.