Parasitology (1975), 70, 203-222 With 3figuresin the text
203
Laboratory studies with some recent anticoccidials JOHN F. RYLEY and ROBERT G. WILSON Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, England (Received 23 August 1974) SUMMARY
The activities of monensin, lasalocid and halofuginone against Eimeria tenella, E. brunetti and E. necatrix have been studied under laboratory conditions. Complete control of experimental infections in the chick, separable from toxicity, was not obtained with monensin, but was achieved with the other two compounds at levels of 150 and 6 ppm in the food respectively. All three compounds appear to inhibit coccidial development very early in the life-cycle, and to have a fairly rapid lethal effect, monensin and lasalocid more so than the febrifugine derivative. In vivo observations have been supplemented with in vitro studies. Some discussion of the difficulties of relating laboratory experiments to field performance is given. INTRODUCTION
Among the antibiotics, two polyether carboxylic acids, monensin and lasalocid (X537A), have been or are being developed as prophylactic anticoccidial agents. Both are ionophores, influencing the permeability of biological membranes to monovalent and divalent cations respectively; lasalocid, by virtue of its influence on Ca2+ ions, appears to have potential therapeutic applications in certain types of heart disease. Although an extensive bibliography is accumulating on both products, most is so far in abstract form or refers to aspects other than anticoccidial activity. The scant literature on anticoccidial activity has been reviewed by Ryley & Betts (1973); Mitrovic & Schildknecht (1974) have subsequent to our work presented some data on the anticoccidial activity of lasalocid. Starting from substances of biological origin, a number of febrifugine derivatives were synthesized some years ago as potential antimalarial agents. One of these, 7bromo-6-chlorofebrifugine (now known as halofuginone), was claimed to have very marked anticoccidial activity (see Ryley & Betts, 1973). After a lapse of several years, this substance is now undergoing field development as an anticoccidial agent (Yvor6, Foure, Aycardi & Bennejean, 1974). In the present paper we present data on the anticoccidial activity of monensin, lasalocid and halofuginone under laboratory conditions, and describe some experiments carried out both in vivo and in vitro which throw light on their mode of action.
204
J. F. RYLEY AND B. G. WILSON
MATERIALS AND METHODS
Monensin has been obtained both in pure form and as a premix (Coban) by courtesy of Mr M. L. Clarke of Eli Lilly & Co. Lasalocid (Avatec) was kindly provided by Dr W. Rehm of Hoffmann-La Roche & Co. Ltd; most of the experiments were carried out with a 20 g sample lot No. 478-145 ARX, but the experiment summarized in Table 7 utilized material of lot No. 2936-94. This product is still under development, and standardization of purity and biological activity from batch to batch has not yet been achieved. Some of the halofuginone was supplied as 90% pure material by Dr E. Waletzky of American Cyanamid; other experiments were carried out using 'Stenoral' premix (Roussel-Uclaf), which contains 0-6% active ingredient in an inactive carrier. 'Lerbek' (Dow), a premix yielding at user level 100 ppm clopidol and 8-35 ppm methyl benzoquate, and robenidine (American Cyanamid) were used for comparative purposes. Biological methods used in the present study have already been fully described (Ryley & Wilson, 1971, 1972). Details of individual experiments are given in the tables, figures or text. Tables indicate days over which observations on mortality, weight gain and oocyst production were made. Day 0 in all cases was the day of inoculation; thus day — 1 represents the day before inoculation, day 3 represents 3 days after inoculation. Treatment 0—3 means that birds were fed medicated food from the time of inoculation until 72 h later, and thereafter unmedicated food; 2-12 means that birds received unmedicated food until 48 h after inoculation, and thereafter until day 12 diet containing drug. 0 -^ in Tables 1, 5 and 8 indicates that medicated food was supplied from the time of inoculation instead of 24 h previously as in the other groups. Blood in droppings was estimated visually on a scale of 0-3 for each cage; the faecal score is the average of all the replicates on the same treatment. RESULTS
Monensin Therapeutic activity Monensin is recommended for field use at an inclusion rate of 121 ppm. Table 1 summarizes cage experiments carried out with four species of coccidia using monensin at five different levels. In experiments of this type we have been in the habit of starting medication on the same day, but a few hours before, inoculation. With a variety of drugs (e.g. methyl benzoquate, robenidine, an azauracil derivative, etc.) we have been able to achieve complete coccidiosis control under such conditions. Not so with monensin. Preliminary experiments indicated that monensin displayed better activity when medication was started 24 h before inoculation, and slightly better still when given from 48 h before inoculation. Table 1 indicates that 121 ppm monensin given from 24 h before inoculation was unable completely to control an E. tenella challenge; some mortality and faecal blood were noted, weight gain was poor and there was a massive oocyst output. Control was even poorer when drug was administered from the time of inoculation only. Although increased incorporation rates of
Recent anticoccidials
205
Table 1. Anticoccidial activity of monensin in the chick E. tenella
E. necatrix
A
A
t
/o
Treatment (ppm drug in feed)
mortality (4-8)
Non-infected control Infected control Monensin 363 Monensin 242 Monensin 121 Monensin 60 Monensin 30 Monensin 121, 0-*
0-0 93-8 2-1 0-0 18-8 64-6 89-6 54-2
Av. wt. /o gain mor( - 1 to Oocysts Faecal tality (5-8) score (4-8) 8) — 0-0 0-9 80-5 — 21-6 2-7 70-8 00 0-0 34-3 0-08 63-8 0-0 2-28 0-0 00 62-3 15-76 1-7 2-7 12-5 28-1 — 21-3 16-7 2-8 43-2 4-78 2-0 250 E. brunetti
Av. wt. gain ( - 1 to Oocysts Faecal (5-8) score 8) — 81-0 00 26-3 30 31-8 0-00 0-0 63-4 0-00 0-0 841 10 0-50 1-07 2-5 551 0-90 3-0 511 0-70 62-3 2-8
E. acervtdina
A
A
1
/o
Treatment (ppm drug in feed)
mortality (4-7)
Non-infected control Infected control Monensin 363 Monensin 242 Monensin 121 Monensin 60 Monensin 30 Monensin 121, 0->
00 68-8 0-0 0-0 0-0 2-1 8-3 16-7
Av. wt. Av. wt. gain gain ( - 1 to Oocysts ( - 1 to Oocysts (4-7) (4-7) 7) V) . 66-0 67-8 3-2 — 12-5 1-76 36-4 301 017 0-90 55-6 461 1-30 0-85 52-4 64-3 7-51 4-71 35-9 47-8 6-81 6-84 29-5 6-04 230 7-61 370 41-8 609 5-87
Forty-eight birds per treatment; inoculated on day 0 with 403 000, 257 000, 250 000 or 4 x 106 oocysts of E. tenella, E. necatrix, E. brunetti and E. acervulina, respectively. Medicated food supplied from day — 1, except where indicated by 0-s-, which indicates medication supplied from time of inoculation only.
monensin resulted in better coccidial control, as indicated by reduced mortality, oocyst output and faecal score, weight gains were poor. Monensin apparently has an appetite-depressing effect, and 121 ppm seems to be a compromise between what the bird will take and what will give adequate anticoccidial activity. Rather better results were achieved with 121 ppm monensin against the other three species, though in all cases there was incomplete control as indicated by an appreciable oocyst output, and in each case better results were achieved when medication was started on the day before rather than the day of inoculation. Restricted medication Table 2 summarizes an experiment in which groups of 56 chicks were inoculated with 617000 oocysts of E. tenella and treated with food containing 242 ppm monensin over the periods indicated. This incorporation rate was chosen in an attempt to achieve maximum control when drug was present. It will be noted, however, that although control - at least as measured by mortality - was achieved
206
J. F. BYLEY AND K. G. WILSON
Table 2. Effect of restricted medication with monensin on chick mortality due to Eimeria tenella Days after inoculation Treatment , (days) 1 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-12 1-12 2-12 3-12 -1-12
— — — — — — — — — — — —
A
2
3
— — — — —
— —
—. — —
—. —
—.
.— .— — •
—
—
—
—
—
—
4
5
6
7
4 5 4 1 2 — — 1 3 2 4 10
33 13 8 3 2 4 5 7 3
8 9 9 6 1 3 — 3 1 1 8 4
3 6 1 — — 1
3 19 20
•
—
1 1 — — —
8
9
10
11
— — —. — — —. —.
— — — — — — —
1 3 1 1 — — 1 —
3 1
3 1 — — — —
. — — —
1
2 —
3
1 — —
, Total 12 deaths — 52 — 37 — 26 — 12 — 5 — 8 — 6 — 12 — 8 — 8 — 33 — 37 — 0
Fifty-six birds per treatment; inoculum, 617000 oocysts; monensin supplied at 242 ppm in diet.
when medication was given from day — 1 to day 12, in all groups where treatment started at the time of inoculation, some birds died from coccidiosis. With shorter periods of treatment, more birds died, but a clear mortality picture failed to emerge. Because E. brunetti seemed to be more susceptible (Table 1), a similar experiment was carried out using groups of 48 chicks inoculated with 190000 oocysts of this species and medicated for restricted periods with food containing 180 ppm monensin. Mortality was minimal, and so the parameters of daily weight and daily oocyst output were followed. Fig. 1 indicates that although continuous medication with monensin did not completely control weight gain, virtually an equivalent growth curve was obtained when drug was administered for the first 24 h of infection only. Likewise a delay in the start of medication of 1-4 days after inoculation improved growth somewhat compared to the infected unmedicated controls. All groups, however, showed some oocyst output, in every case reaching a maximum on days 6-7 (Table 3). Histological experiments Groups of 10-day-old chicks were fed diets containing 363, 242 or 121 ppm monensin and 3 days later were inoculated with 10 million oocysts of E. tenella or E. brunetti; control birds on unmedicated food were given 10 million, 200000 or 5000 oocysts. Two birds in each group were killed each day, and four pieces of caecum or five pieces of small intestine were taken, washed through with saline, fixed in Stieve's fluid (water, 570 ml; HgCl2, 34-5 g; 40% formaldehyde, 150 ml; glacial acetic acid, 30 ml), sectioned and stained with haematoxylin and eosin or PAS-AO. In this type of experiment, it was necessary to use very large inocula of oocysts in order to get enough first-generation stages to make microscopical observation
Recent anticoccidials 140
207
Non-infect. control 0-1 0-9
(A) 120
Non-infect. control 0-9
100 Infect, control
80
60 4 Days
4 Days
8
Fig. 1. Effect of restricted medication with monensin on growth of chicks infected with Eimeria brunetti. Forty-eight chicks per treatment, inoculated on day 0 with 190000 oocysts; diet containing 180 ppm monensin supplied over periods indicated.
Table 3. Oocyst output during restricted medication with monensin (same experiment as in Fig. 1 using Eimeria brunetti) Oocyst output (millions per chick) Treatment days
5-6
6-7
7-8
8-9
Total
Control 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-14 1-14 2-14 3-14 4-14 5-14 1-5
0-73 1-40 0-96 0-30 0-35 0-20 0-38 0-32 0-24 1-79 1-46 1-99 1-46 0-98 1-70
2-08 2-89 3-31 210 1-56 1-33 0-87 114 0-90 3-32 2-95 2-78 2-38 2-29 4-37
1-51 2-42 1-34 1-49 1-41 1-02 0-56 0-48 0-42 1-42 1-23 1-29 117 1-08 3-57
115 1-82 0-99 101 1-14 0-61 012 009 0-10 0-20 0-29 0-23 0-20 0-34 1-30
5-47 8-53 6-60 4-90 4-46 316 1-93 203 1-66 6-73 5-93 6-29 5-21 4-69 10-94
of the effects of drug on these stages possible. With E, tenella at the three drug levels tested, haemorrhage was noted in each case, but caecal core formation only at 121 ppm. At all three levels, all stages of the life-cycle could be observed microscopically at appropriate times, but there was an overall delay of up to 24 h in the development of each stage, and an overall appreciable reduction in the number of developing parasites when the size of the inoculum was taken into account. With 363 ppm monensin, some of the mature second-generation schizonts observed on days 4 or 5 contained abnormal rounded-off merozoites similar to those seen with lasalocid. In the case of individual parasites which failed to develop, inhibition appeared to be right at the start of the life-cycle; there was no accumulation of partially developed but inhibited parasites as has been observed with some drugs, e.g. robenidine (Ryley & Wilson, 1971). Similar
208
J. I\ EYLEY AND E. G. WILSON
effects were noted with E. brunetti; some parasites developed to produce sexual stages, but there was an appreciable reduction in the numbers so doing, particularly at 242 and 363 ppm. Tissue culture experiments Complete inhibition of E. tenella and E. brunetti in chick kidney tissue culture occurred with 0-002 ppm monensin. When 0-005 ppm drug was added to cultures inoculated with sporozoites of E. tenella, the parasites were inhibited as multinucleate first-generation schizonts. Vacuolation and degeneration were evident from 48 h, and little recognizable parasite material was found at 66 h. When cultures containing 0-005 ppm monensin were inoculated with sporozoites of E. brunetti, very few parasites developed beyond the trophozoite stage before undergoing degeneration; first-generation development of E. brunetti in culture is slower than E. tenella, and inhibition therefore set in at an earlier stage. No second-generation development was seen when cultures inoculated with E. tenella and containing 0-005 ppm monensin were washed free of drug 24, 48 or 72 h after inoculation. A few mature first-generation schizonts were seen subsequent to 24 h withdrawal, but these did not give rise to second-generation schizonts. When 0-005 ppm drug was added 24, 48 or 72 h after inoculation, inhibited parasites started to degenerate within 24 h of drug addition. When drug was added 96 h after inoculation, some second-generation schizonts matured, but the merozoites rounded off after release; late multinucleate second-generation schizonts became disrupted and vacuolated. When cultures were treated with 0-005 ppm drug for 24, 48 or 72 h periods starting 24 or 48 h after inoculation, no mature second-generation schizonts developed. However, treatment during the period 72-96 h allowed some schizonts to mature and release their merozoites apparently normally, while other merozoites became rounded and multinucleate schizonts vacuolated. When cell cultures were treated with 0-005 ppm monensin for 24 or 48 h and washed free of drug immediately before inoculation with E. tenella, no inhibitory effect was seen on either cell or parasite development. When, however, the preinoculation drug level was raised to 0-025 ppm (10 times the minimum active level) very few or no second-generation schizonts subsequently developed, although cell cultures appeared to be growing normally. Kidney cell cultures were grown for 24 or 48 h in medium containing 0-025 ppm drug and then allowed to continue growth in drug-free medium for periods up to 72 h before inoculation with sporozoites. Table 4 summarizes the effects on parasite development subsequently observed. Lasalocid Therapeutic activity Table 5 summarizes experiments carried out to determine dose-response relationships of lasalocid using three species of coccidia. Lasalocid at 75 ppm showed some activity under our experimental conditions, but did not give adequate control in the case of E. tenella or E. necatrix; with E. brunetti, although weight gain and mortality were controlled, there was a small oocyst output. In all cases,
Recent anticoccidials
209
Table 4. Effects of the preincubation of tissue culture cells with monensin on the subsequent development of Eimeria tenella Drug on
Drug off Inoculated
(h)
(h)
(h)
0 0
24 24
48 72
24
96
48 48 48
72 96 120
0 0 0
Parasite development Inhibited as multinucleate primary schizonts Inhibited as multinucleate primary schizonts; few mature schizonts produced, but no subsequent secondary development Some early secondary schizonts present at 168 h, but no mature forms developed subsequently Inhibited as multinucleate primary schizonts Inhibited as multinucleate primary schizonts Early secondary schizonts present at 192 h; no corresponding mature development seen at 216 h. Traces of mature secondary development at 240 h
150 ppm of the material used produced excellent results. Lasalocid at 75 ppm administered from the time of inoculation rather than 24 h previously produced approximately the same result; certainly there was not the marked falling-off in control observed with monensin. Lerbek and robenidine, at recommended levels, gave very good control with all species; monensin once again was rather disappointing, particularly with E. tenella. Restricted medication Table 6 summarizes an experiment carried out to indicate the stage in the life-cycle at which lasalocid acts. Although a level of 75 ppm is being recommended for field use (Mitrovic & Schildknecht, 1973, 1974), Table 5 indicates that at this level under our experimental conditions, incomplete control of the infections was achieved. Since 150 ppm of this batch of material did on the other hand give complete control, including elimination of oocyst production, this level was chosen for mode of action studies. As Table 6 indicates, 47/48 untreated birds died, the majority doing so on day 5. Treatment restricted to the first 24 h of the infection reduced mortality below 50%, and again the majority of birds which died did so on day 5; treatment for 2 or more days resulted in little mortality. Growth curves based on daily weighings (Fig. 2 A) show a similar picture; medication for the first day of the infection only gave partial control of the inhibitory effects of coccidia on growth, while medication for 2 or more days was adequate to give growth curves indistinguishable from the uninfected controls. Daily oocyst counts were also carried out on each group; Table 6 indicates total oocyst production over days 5-12 in each group expressed as millions of oocysts per chick. Medication for the first 4-5 days was sufficient to prevent almost completely oocyst production on drug withdrawal; no displacement of the day of maximum oocyst production was observed with shorter treatments. These experiments suggest that lasalocid exerts an initial coccidiostatic action at 150 ppm, but that with continued contact, this effect quickly becomes
Av. wt. /o gain mortality Oocysts (4-8) ( - 1 to 8) (5-8) . . 82-8 0-0 — 16-1 95-3 79-3 0-00 0-0 62-6 14-45 12-5 — 21-7 67-2 — 141 95-3 — 95-3 13-5 36-4 10-30 15-6 37-7 12-27 391 9-00 239 62-5 0-00 75-8 0-0 000 79-5 0-0 0-0 30 0-4 2-4 30 3-0 30 2-8 2-9 30 0-0 0-0
0-0 92-2 0-0 31 35-9 78-1 89-1 9-4 00 42-9 00 0-0 83-0 18-7 83-5 71-0 40-4 16-3 17-5 54-0 79-1 39-5 80-2 83-5 0-00 0-58 0-57 — — 0-47 0-65 0-41 0-02 0-00
Av. wt. /o Faecal mortality gain Oocysts score (4-8) ( - 1 to 8) (6-8)
A
0/
E. necatrix
A
^
E. tenella 0/
A
E. brunetti
0-0 2-8 0-0 0-1 1-5 1-8 2-6 1-3 01 2-1 00 0-0
1-30 000 0-00
67-7 19-4 71-8 71-9 690 47-8 32-7 70-6 70-6 70-3
0-0 20-3 0-0 00 00 0-0 6-3 1-6 .— 00 00
4-51 000 0-15 504 11-95 9-70
Av. wt. /o Faecal mortality Oocysts gain (4-7) (-Ito7) score (4-7)
i
Sixty-four birds per treatment; inoculated on day 0 with 646000, 222000 or 197000 oocysts of E. tenella, E. necatrix and E. brunetti, respectively. Medicated food supplied from day — 1, except where indicated by 0 ->•, which means medication supplied from time of inoculation only.
Non-infected control Infected control Lasalocid 150 Lasalocid 75 Lasalocid 37-5 Lasalocid 19 Lasalocid 9 Lasalocid 75, 0 -> Monensin 121 Monensin 121, 0 -> Lerbek 108 Robenidine 33
Treatment (ppm drug in feed)
t
Table 5. Anticoccidial activity of lasalocid in the chick
o
CO
w o
t)
H
w
o
Recent anticoccidials
211
Table 6. Effect of restricted medication with lasalocid on chick mortality due to Eimeria tenella Treatment days — 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-12 1-12 2-12 3-12
Days after inoculation , • —-*• 1 2 3 4 5 6 7 8 9 1 0 11 — — — 1 4 3 3 _ _ _ _ _ _ — — — — 14 3 2 1 1 — — _ _ _ _ _ i i _ _ _ _ _ _ _ _ _ _ _ _ i _ _ _ _ _ _ — _ — _ 1 — — — _ _ _ _ _ _ _ _ _ _ _ — — — — — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 1 — — — _ — _ _ _ _ 2 5 1 1 1 — — — _ _ _ 1 3 5 1 1 i _ _ _
Oocyst , Total production 12 deaths 5-12 47 — — 21 23-87 2 11-43 _ l 4-81 — 1 0-94 _ o 0-17 o 0-01 — o 0-00 o 0-00 — 3 0-75 — 28 1-31 _ 38 019
Forty-eight birds per t r e a t m e n t ; inoculum, 430000 oocysts; lasalocid supplied a t 150 p p m i n diet. 120
r
Non-infect. control
•
100
Non-infect. control
0-1
80
Infect, control
Infect, control
60 I
1
1
6 8 8 0 2 4 Days Days Fig. 2. Effect of restricted medication with lasalocid on growth of chicks infected with Eimeria tenella. Details as recorded in Table 6.
0
2
4
6
coccidiocidal; 2 day's treatment was sufficient to kill the majority of the coccidia as judged by mortality and weight gain control following drug withdrawal, while around 5 day's treatment was required to give a complete coccidiocidal effect as judged by oocyst production following drug withdrawal. The mortality pattern observed and the fact that medication had to start within 24 h of inoculation to prevent mortality (Table 6) suggests that lasalocid exerts its action somewhere during the first asexual cycle of reproduction. Table 7 summarizes an experiment carried out with E. tenella to examine the effects of lasalocid against later stages of the life-cycle. The first attempt to do this showed that 150 ppm of the new batch of lasalocid did not completely prevent oocyst production when given continuously from the time of inoculation; the table records daily oocyst counts carried out on groups of six chicks inoculated with 2000 oocysts on day 0 and given food medicated with 300 ppm lasalocid from the times indicated until the end of the experiment. It will be seen that if medication was started within 72 h of inoculation, no oocysts were produced; 14
P A R 70
212
J. F. RYLEY AND R. G. WILSON
Table 7. Effect of delayed treatment with lasalocid on the oocyst output of chickens infected with Eimeria tenella (Figures are oocyst outputs in millions/chick/day) Days postinfection
Period of treatment (days) Infected control
4-5 5-6 6-7 7-8 8-9
0-08 3-20 11-38 5-44
9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18
1-47 1-64 0-49 1-77 1-32 5-82 7-37 3-47 2-53
Total
50-09
411
A
0-18
1-18
2-18
3-18
4-18
5-18
6-18
7-18
000
0-00 0-00
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00
0-00 0-00 0-00
0-05 0-49 0-20
0-08 2-67
0-08 2-89 1111
000
009 001
3-26 1-16 0-16 0-02 0-01 0-00
0-06 1-93 7-71 5-25 2-30 0-89 0-37 0-04 0-05 0-00
0-00 0-00
000
000
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00
0-00
0-00
000 000
0-00 000 000
0-00 000
0-00 0-00 0-00
000 000
0-00 0-00 0-00
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 000
0-00
0-00 0-00 000
0-00 0-00 0-00 0-00 0-00 0-00 0-84
101
000 000
814
2-91 0-47 0-22 0-36 0-03 0-01
000
000
000
0-00 0-07 0-02
8-37
18-69
0-00 0-00 0-00 26-22
0-00 0-00
when medication was started on day 4, few oocysts were subsequently produced, and even when medication was delayed until the infection had become patent, oocyst output was fairly quickly stopped. Histological experiments Chicks were inoculated with 10 oocysts of E. tenella and fed a diet containing 150 ppm lasalocid beginning at the time of inoculation or 24 or 48 h later. Two birds from each group were killed each day, and two pieces of each caecum taken for histology, being processed as before. When drug was present from the time of inoculation, both treated and control sections showed undeveloped intracellular parasites at 24 h. At 48 h, control sections contained appreciable numbers of medium and large multinucleate first-generation schizonts, and a few mature schizonts. Sections from treated birds showed far fewer parasites, and those that were present showed retarded development; some undeveloped sporozoites/ trophozoites were found, and a few small multinucleate and the occasional larger schizont were seen, mostly showing signs of vacuolation and degeneration. A few degenerating medium-sized first-generation schizonts were found at 72 h in treated birds, but no parasites could be found at 96 h; there was no evidence of tissue destruction or bleeding. By contrast, control birds showed massive bleeding and tissue destruction by 72 h (due to break-up of the first-generation schizonts produced from the very heavy inoculum) and countless developing second-generation schizonts, many of which were mature by 96 h. When medication was delayed until 24 h after inoculation, there was a retardation and reduction but not complete suppression of first-generation schizogony, and likewise an overall reduction in, but far from complete suppression of, second-
Recent anticoccidials
213
generation schizogony; bleeding and tissue destruction were evident as a consequence of second-generation schizogony, but many apparently mature schizonts contained rounded-off bodies rather than elongate merozoites. Subsequent experiments with a lower inoculum (5000 oocysts) indicated a very marked reduction in sexual development in birds where medication was started 24 h after inoculation. A 48 h delay in medication produced little evidence of drug activity at the histological level - in spite of the results recorded in Table 7 on eventual oocyst production. Tissue culture experiments No second-generation development was observed in cultures inoculated with E. tenella or E. brunetti and treated with 0-004 ppm lasalocid. When 0-01 ppm drug was added to cultures from the time of inoculation with E. tenella, some mature first-generation schizonts could be found from 48 h on. There were, however, fewer schizonts than in untreated cultures, and in cultures where merozoites were released and invaded fresh kidney cells, no further development was noted. With higher levels of drug, fewer schizonts matured; many schizonts were inhibited before or at merozoite formation. By contrast, addition of 0-01 ppm lasalocid to cultures along with sporozoites of E. brunetti resulted in inhibition of the majority of parasites at the sporozoite or trophozoite stage; a few small multinucleate first-generation schizonts together with the occasional mature schizont could be found at 64 h, but no further development was seen. Cultures containing 0-01 ppm lasalocid were inoculated with E. tenella, and the drug removed after various intervals. When drug was removed after 24 or 48 h, parasites were subsequently able to complete two asexual cycles of development, although the number of second-generation schizonts in the latter case was small; hardly any second-generation parasites were found when drug had been present for 72 h. Treatment with 0-01 ppm drug for a 24 h period during days 1-4 caused a delay in development, but many second-generation schizonts developed after drug withdrawal. Fewer second-generation schizonts developed when the cultures were treated for 48 h, and hardly any when the period of treatment was extended to 72 h. In cases where the limited treatment started 48 or 72 h after inoculation, some of the second-generation merozoites became rounded off before or after rupture of the schizont. No second-generation schizonts developed when 0-01 ppm drug was added 24 or 48 h after inoculation; first-generation schizonts matured and released their merozoites, but no further development was seen. When drug was added 72 or 96 h after inoculation, many normal second-generation schizonts developed, but others failed to produce normal merozoites, rounded-off bodies being released instead. No effect on parasite development was seen when cultures were pretreated with up to 0-1 ppm lasalocid (25 times the minimum effective level) for 48 h and then washed free of drug immediately before inoculation with sporozoites.
14-2
0-0 100-0 0-0 0-0 0-0 7-8 92-2 0-0 32-8 53-1 0-0 0-0
80-1 23-8 58-4 76-0 81-7 63-3 21-5 80-1 54-8 39-2 79-1 81-4 0-00 0-00 0-14 9-94 — 0-08 11-00 2-96 0-00 0-00
E. necalrix
00 2-1 0-0 0-0 0-0 1-3 2-5 00 2-5 2-5 0-0 00 0-0 92-2 0-0 0-0 5-2 500 921 8-1 111 60-7 0-0 00
66-4 9-6 52-8 62-3 59-6 25-8 10-8 65-4 53-0 30-7 67-9 63-9 . 0-00 0-00 0-01 0-11 — 0-05 0-52 0-66 0-00 0-00
Av. wt. /o Faecal mortality gain Oocysts score (4-8) ( - 1 to 8) (5-8)
t
A
0-0 30 00 0-0 0-3 2-5 2-9 0-4 1-6 1-9 0-0 0-0
0-0 31-3 0-0 0-0 00 10-9 10-9 0-0 0-0 1-6 0-0 0-0
66-8 13-6 47-9 60-9 57-8 29-8 19-5 561 58-6 43-4 60-7 61-8
Av. wt. /o gain Faecal mortality (4-7) ( - I t o 7 ) score 0/
E. bruneth
3-77 0-00 000 0-37 3-27 4-36 019 3-31 5-75 0-00 000
Oocysts (4-7)
Sixty-four birds per treatment; inoculated on day 0 with 319000, 250000 or 298000 oocysts of E. tenella, E. necatrix and E. brunetti, respectively. Medicated food supplied from day — 1, except where indicated by 0 ->, which means medication was supplied from the time of inoculation only.
Non-infected control Infected control Halofuginone 12 Halofuginone 6 Halofuginone 3 Halofuginone 1-5 Halofuginone 0-75 Halofuginone 3, 0 -*• Monensin 121 Monensin 121, 0 -> Lerbek 108 Robenidine 33
Treatment (ppm drug in feed)
0/
Av. wt. /o mortality Oocysts gain (5-8) (4-8) ( - 1 to 8)
A
E. tenella
Table 8. Anticoccidial activity of halofuginone. in the chick
O
m
P
S3
>
F
to
Recent anticoccidials
215 Non-infect. control 2-14
Non-infect. control 0-5 0-4 0-3
140 120
3-14
0-2
100
0-1 80
Infect, control
Infect, control
60 I
4 Days
4 Days
Fig. 3. Effect of restricted medication with halofuginone on growth of chicks infected with Eimeria tenella. Details as recorded in Table 9.
Halofuginone Therapeutic activity Halofuginone has been evaluated at five inclusion rates against three species of coccidia (Table 8). Complete control of mortality and oocyst production was achieved with 6 ppm, and quite good control with 3 ppm. It should be noted that control dropped markedly when the inclusion rate was reduced below 3 ppm, and that weight gains were not as good as the controls at 6 ppm and markedly reduced at 12 ppm, indicating toxicity. Once again, excellent control of all species was achieved with Lerbek and robenidine, and only partial control by monensin. Unlike the situation with monensin, medication with halofuginone could be started at the time of inoculation and still produce satisfactory results. Restricted medication
Therapeutic experiments (Table 8) indicated that 3 ppm just about gave control of the coccidial challenges used, but in view of the rather steep doseresponse curve with halofuginone, levels of 4-5 and 6 ppm have been used in some of the mode of action studies hopefully to obtain more decisive effects. Table 9 and Fig. 3 summarize an experiment carried out using 4-5 ppm halofuginone in restricted schedules of medication. Oocyst production data (Table 9) indicates that in this particular experiment, complete suppression was not achieved by continuous medication, although no birds died and there was little evidence of infection from growth data. That the drug was not immediately coccidiocidal under these conditions is apparent from the relapses indicated by mortality and oocyst production following drug withdrawal after administration for 1-5 days; growth curves gave a rather less sensitive indication of relapse. To avoid mortality, drug treatment had to be started within 24 h of infection, although a delay of 48 h resulted in but few deaths and a growth curve indistinguishable from that produced under continuous medication. An experiment was carried out to investigate the effects of halofuginone at 6 ppm against the later stages of the life-cycle of E. tenella concurrently with
0-4 0-5 0-7 0-10 0-14 1-14 2-14
Treatment days 8 19 7 3 — 1 — 7 6 1 .— 1
— — 13 2 — —
•— 1 2 9 .— —
— — 2 12 10 — ,
10 — — — — 7 2 ,
11 — .— .— .— 1 5 .
12 — — — — — 4 1
13
14
Sixty-four birds per treatment; inoculum, 409000 oocysts; halofuginone supplied at 4-5 ppm in diet.
2 26
54 24 1 1 — —
Days after inoculation
63 51 31 28 18 13 2 0 0 0 3 32
— 43-20 45-24 53-05 35-20 3-57 2-27 2-29 215 2-66 6-06
Oocyst Total production 5-14 deaths
Table 9. Effect of restricted medication with halofuginone on chick mortality due to Eimeria tenella
to
CF O Q
M
?
o w
>
M
K|
OS
Recent anticoccidials
217
Table 10. Effect of delayed treatment with halofuginone on the oocyst output of chickens infected with Eimeria tenella (Figures are ooeyst outputs in millions/chick/day) Days postinfection 4-5 5-6 6-7 7-8 8-9
9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18 Total
Period of treatment (days) Infected control 0-09 2-55 11-48 5-90 4-34 2-96 2-43 1-79 0-53 0-66 4-25 8-83 8-91 3-80 58-52
A
, 0-18
1-18
2-18
3-18
4-18
5-18
6-18
7-18
0-00
0-00 0-00 0-00 0-01
000
0-00 0-00 0-02 0-03
0-05 0-49
0-07 0-95 0-41 1-34 0-19
0-08
0-08 2-67 12-09 8-68 6-77 2-29 0-67 0-20 0-01 0-01
000
0-00 0-00 001 000 000
0-00 000 000
0-00 0-00 0-00 0-00 001
000
0-00 0-00 0-00 0-00 000
0-00 0-00 0-00 0-00 0-01
0-00 0-08 0-11 0-02 0-00 0-00 000 000
000
0-00 0-00 0-02 0-00 0-00 0-00 0-00
0-00 0-00 0-00 0-00 0-00
000 000
0-21
0-07
006
0-06 0-00 0-00 0-00 000
0-00 000 000
0-00 0-00 0-00 0-66
003
0-00 0-00 000 000
0-00 0-00 000
0-00 2-99
216
8-25 5-30 1-66 0-23 0-04 000 000
0-00 0-00 0-00 0-00 0-00 17-72
003
0-00 000
0-00 33-50
that recorded for lasalocid in Table 6. As Table 10 indicates, oocyst outputs were negligible when medication was started at any time up to 96 h after inoculation; medication started once the infection had become patent resulted in a fairly rapid cessation of oocyst production and a reduction iri total output compared with the control. Histological experiments Chickens inoculated with 107 oocysts of E. tenella were treated in groups with diet containing 6 ppm halofuginone starting at the time of inoculation or 24 or 48 h later. Two birds from each group were killed each day and two pieces of tissue taken from each caecum for histology. Examination of slides stained with haematoxylin and eosin or PAS-AO indicated that when halofuginone was present from the time of inoculation, no coccidial development took place; parasites invaded crypt epithelial cells, a parasitophorous vacuole was formed, but the parasites rounded off, degenerated, and were quickly eliminated. When medication started 24 h after inoculation, some small and medium-sized first-generation schizonts were seen at 48 h, some of which appeared to be vacuolated and degenerating. At 72 h there were some vestigial remains of degenerating first-generation parasites, but no trace of second-generation parasites; sections were clear of coccidia by 96 h. When drug administration was delayed until 48 h after inoculation, no consistent anticoccidial effect could be discerned at the histological level. In a repeat experiment involving 3 ppm drug, most of the parasites failed to develop at all when drug was present from the time of inoculation, although a few underwent the complete life-cycle with a 24 h delay at each stage. Following a 24 h delay in the start of medication with 3 ppm drug, plenty of parasites could
218
J. F. RYLEY AND R. G. WILSON
be found at each stage, though there was obviously an overall reduction in numbers in view of the immense inoculum. No effect could be noted histologically when drug treatment was started 2 or 3 days after inoculation - although the experiment recorded in Table 10 indicates the ability of late treatment to suppress oocyst production. Tissue culture experiments The drug showed complete activity against E. tenella at 0-01 ppm. Incomplete and variable activity was achieved with 0-005 or 0-002 ppm, and no activity with 0-001 ppm. Continuous treatment with 0-01 ppm drug prevented any development beyond the sporozoite stage. Similar levels of drug showed similar effects with E. brunetti. When drug (0-01 ppm) was removed 24 h after inoculation, numerous mature second-generation schizonts could be found 5 days later; fewer parasites developed following drug withdrawal at 48 h, and none when drug was withdrawn at 72 h. When cultures were treated with 0-01 ppm drug for 24 h starting 24, 48 or 72 h after inoculation, all produced mature second-generation schizonts 3-5 days after drug withdrawal. If cultures were treated for 48 h, starting 24 or 48 h after inoculation, only a limited amount of second-generation schizogony was produced after drug withdrawal. When cultures were treated over the period 24-96 h after inoculation, only a few multinucleate first-generation schizonts had developed 3 days after withdrawal, and no second-generation development took place. Starting treatment with 0-01 ppm drug 24, 48, 72 or 96 h after inoculation showed that the drug was active against life-cycle stages other than the sporozoite. Administration commencing 24 h after inoculation resulted in inhibition at the sporozoite or trophozoite stage. Following addition at 48 h, immature firstgeneration schizonts failed to mature, but became vacuolated and degenerate; mature first-generation schizonts present at the time of drug addition failed to develop further. When drug was added at 72 h, early second-generation schizonts became vacuolated and degenerate. Similar effects were produced when drug treatment was started at 96 h, although mature forms present produced merozoites and subsequently released them. Development of drug resistance
Eight passages of E. tenella have been carried out in groups of three, 3-week-old chicks reared in plastic-film isolators, in the presence of increasing levels of halofuginone. In the initial passage, which involved an inoculum of 106 oocysts and a drug level of 3 ppm, faecal blood and caecal cores were produced. The total oocyst yield from this passage was inoculated into further chicks given food containing 6 ppm halofuginone; blood and caecal cores were again produced. Five further passages have been achieved in the presence of 12 ppm halofuginone followed by one with 18 ppm. Moderate numbers of oocysts were obtained at each passage, but no blood or caecal cores were noticed. The coccidia developing in the presence of 18 ppm halofuginone appear to be E. acervulina, presumably derived from an unsuspected slight contaminant in the original culture!
Recent anticoccidials
219
DISCUSSION
Under the experimental conditions reported here, Lerbek and robenidine gave very good control of the three species of coccidia tested. Over the past 30 years there has been a succession of anticoccidial agents developed, each giving better control under experimental conditions, often at a lower absolute dose, than its predecessors. It is hard to imagine how a new drug could be launched which did not appear better than currently available anticoccidials. Although few details of its anticoccidial activity have been published (see Ryley & Betts, 1973), monensin, currently the most commonly used anticoccidial agent in the U.S.A. is not very impressive by comparison with the other drugs in stringent laboratory tests. In the present studies it compares very unfavourably with Lerbek, robenidine, lasalocid or halofuginone, yet it is undoubtedly very successful commercially. This fact causes concern to the experimental coccidiologist who wonders what criteria of activity in the laboratory to apply to a compound in order to decide to take it forward to field trial. Under experimental conditions, monensin seems to require time to build up tissue levels adequate to show maximum activity. Although this of course is of no consequence under field conditions as a prophylactic agent, it does make it difficult to devise satisfactory experimental studies on its point of action. At an incorporation rate of 121 ppm - a compromise between activity and toxicity and/or appetite depression - it does not prevent development of all the parasites in a coccidial challenge. One must, therefore, assume that under field conditions severe challenges are seldom met, and that the drug is able to deal with the actual challenge sufficiently to prevent clinical coccidiosis. If some coccidial development does take place in the presence of drug, presumably adequate immunity develops to prevent later breakdown as the coccidial population in the environment increases (see Reid, Kowalski & Rice, 1972). Our experimental rations are deficient in vitamin K; although a commercial ration containing adequate vitamin K will enable monensin to give better control of the haemorrhagic species in the field, it will not alter the relative efficacies of the various drugs. Jeffers (1974) has published a fascinating study of 308 strains of E. tenella isolated from 1145 litter samples obtained from all the major broiler-producing regions in the USA. Using a somewhat crude method of evaluation, he found, among other things, that the majority of strains isolated from enterprises using 'efficient' anticoccidial drugs, such as Amprol Plus, clopidol, buquinolate or decoquinate, showed resistance to the particular drug being used. Although enterprises using monensin yielded an above average number of isolates of E. tenella (37-8%), none of these showed a reduced sensitivity to the drug. It should be remembered that monensin has not been in use for as long a period of time as the other drugs where resistance has emerged. It is possible that organisms find it difficult to develop resistance against compounds such as monensin, which are interfering with ion transport through membranes, rather than an enzymic reaction which could be by-passed. The possibility also exists that monensin does not act directly on the parasite at all, but rather modifies
220
J. F. RYLEY AND R. G. WILSON
the host cell in some way, making the environment unsuitable for parasitization — another situation in which it would be difficult to visualize the development of drug-resistance. In vitro experiments in which cultures were incubated with monensin and then washed free from drug prior to inoculation with parasites suggest, however, that indirect activity through the host cell probably does not occur. The fact that high concentrations of monensin have to be used to produce antiparasitic effects suggests that drug is absorbed and bound by the cell in a form, which although not removed by medium changing, is able to inhibit parasite development. Dilution of bound drug consequent on repeated cell division would explain the decreasing effects found after increasing intervals between drug removal and inoculation. I t could well be that under field conditions where cycling of coccidia is taking place in the presence of monensin, an inherent limitation in resistance development coupled with an effectively low drug pressure prevents the emergence of drug-resistant lines. Lasalocid is an ionophore bearing some relationship to monensin. In the present studies we were able to achieve complete control of three species of coccidia with 150 ppm drug. Insufficient material was available to determine the maximum tolerated level, but the manufacturer indicates that this depends on purity, which varies from batch to batch. It is apparent, however, that there is a greater separation of activity and toxicity than is the case with monensin, and it will be most interesting in the light of the above discussion to see whether lasalocid, superior to monensin in these laboratory experiments, is more or less successful than monensin under field conditions. Halofuginone is weight for weight one of the most active anticoccidials known. In our experience it shows a steeper dose-response curve than most, but the separation of activity from toxicity is minimal; our observations are in general similar to those recently reported by Yvore et al. (1974). It will be interesting to see how it behaves under field conditions, since the achievement of uniform incorporation rates at such a low level is difficult, and there is so little room for manoeuvre in the acceptable level. As a potentially 'efficient' anticoccidial, and in view of the limited laboratory work on resistance reported, it will be most interesting to note the speed of development of drug resistance under field conditions. The experiments described with monensin illustrate the difficulties which may be encountered when trying to pin-point the stage of the life-cycle at which an anticoccidial drug acts. Restricted medication experiments drew attention to the speed of action of the drug once adequate concentrations had been built up in the host, but did not indicate conclusively the point of action; in birds which managed to survive, very short periods of treatment were as effective as continuous medication. Likewise histological studies were difficult to interpret as a proportion of the parasites were able to complete the life-cycle. Nevertheless, the impression was gained that if an individual parasite was going to be inhibited, it would be inhibited right at the start of the parasitic phase. In vitro experiments enabled studies to be carried out under conditions where complete inhibition, uncomplicated by host toxicity, was possible. Such investigations underlined the
Recent anticoccidials
221
potentially rapid lethal action of monensin, but with E. tenella, drug action was sufficiently slow to allow some first-generation nuclear division to occur. With lasalocid it was necessary to use drug concentrations double that which may be recommended for field use in order to achieve complete interruption of the life-cycle. With 150 ppm drug, it was possible to demonstrate a fairly rapid lethal action on the parasite, and to indicate that under normal conditions of continuous administration, inhibition would occur very early in the endogenous cycle. Histological experiments showed that some parasites could undergo partial development, but few progressed further than halfway through the first asexual phase; slightly more development was possible in vitro, but in neither case did any second generation development occur. In vitro and in vivo experiments indicated that some relapse of development was possible following very restricted periods of treatment, but that in general all stages of the parasite were susceptible to lasalocid, although possibly not quite as sensitive as the very first intracellular forms. It must be emphasized that interesting though the sensitivity of later stages to a drug may be, the only observations of real significance are what happens when the drug is there from the start; in the field drug is continuously administered, and - failing a breakdown in the feeding regimen for any reason — is present when a coccidial challenge occurs. With halofuginone, possibly elevated drug levels had likewise to be used effectively to define the point of drug action. Once again it was the very earliest intracellular stage which was susceptible, but the drug appeared to be somewhat slower in producing a killing effect in that relapses could occur if medication was withdrawn within 5 days. In the experiments reported, attempts have been made to obtain more useful information from restricted medication experiments by daily weighings of birds and daily oocyst counts in addition to the mortality pattern previously used alone (Ryley, 1967; Ryley & Wilson, 1971). The additional information in fact obtained was of limited value, although we have found that with drugs having a pronounced coccidiostatic rather than coccidiocidal effect, e.g. robenidine, such observations can be very informative. We are grateful to Mrs Judith Hazlehurst, Mrs Thelma Robinson and Miss Linda Hardman for invaluable technical assistance.
REFERENCES
T. K. (1974). Eimeria tenella: Incidence, distribution, and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Diseases 18, 74-84. MITBOVIC, M. & SCHILDKNECHT, E. G. (1973). Anticoccidial activity of antibiotic X-537A in chickens. Poultry Science 52, 2065. MITBOVIC, M. & SCHILDKNECHT, E. G. (1974). Anticoccidial activity of Lasolocid (X-537A) in chicks. Poultry Science 53, 1448-55. REID, W. M., KOWALSKI, L. & BICE, J. (1972). Anticoccidial activity of monensin in floorpen experiments. Poultry Science 51, 139-46. RYLEY, J. F. (1967). Studies on the mode of action of quinolone and pyridone coccidiostats. JEFFEBS,
Journal of Parasitology 53, 1151-60.
222
J. F. RYLEY AND R. G. WILSON
RYLEY, J. F. & BETTS, M. J. (1973). Chemotherapy of chicken coccidiosis. In Advances in Pharmacology and Chemotherapy, vol. xi (ed. S. Garattini, A. Goldin, F. Hawking and I. J. Kopin), pp. 221-93. New York, London: Academic Press. RYLEY, J. F. & WILSON, R. G. (1971). Studies on the mode of action of the coccidiostat robenidene. Zeitschrift fur Parasitenkunde 37, 85-93. RYLEY, J . F . & WILSON, R. G. (1972). Comparative studies with anticoccidials and three species of chicken coccidia in vivo and in vitro. Journal of Parasitology 58, 664-8. YVORE, P., FOUBE, N., AYCARDI, J. & BENNEJEAN, G. (1974). Efficacite du Stenorol
(RU 19110) dans la chimioprophylaxie des coccidioses aviaires. Recueil de Medecine Veterinaire 150, 495-503.
Printed in Great Britain
203
Laboratory studies with some recent anticoccidials JOHN F. RYLEY and ROBERT G. WILSON Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, England (Received 23 August 1974) SUMMARY
The activities of monensin, lasalocid and halofuginone against Eimeria tenella, E. brunetti and E. necatrix have been studied under laboratory conditions. Complete control of experimental infections in the chick, separable from toxicity, was not obtained with monensin, but was achieved with the other two compounds at levels of 150 and 6 ppm in the food respectively. All three compounds appear to inhibit coccidial development very early in the life-cycle, and to have a fairly rapid lethal effect, monensin and lasalocid more so than the febrifugine derivative. In vivo observations have been supplemented with in vitro studies. Some discussion of the difficulties of relating laboratory experiments to field performance is given. INTRODUCTION
Among the antibiotics, two polyether carboxylic acids, monensin and lasalocid (X537A), have been or are being developed as prophylactic anticoccidial agents. Both are ionophores, influencing the permeability of biological membranes to monovalent and divalent cations respectively; lasalocid, by virtue of its influence on Ca2+ ions, appears to have potential therapeutic applications in certain types of heart disease. Although an extensive bibliography is accumulating on both products, most is so far in abstract form or refers to aspects other than anticoccidial activity. The scant literature on anticoccidial activity has been reviewed by Ryley & Betts (1973); Mitrovic & Schildknecht (1974) have subsequent to our work presented some data on the anticoccidial activity of lasalocid. Starting from substances of biological origin, a number of febrifugine derivatives were synthesized some years ago as potential antimalarial agents. One of these, 7bromo-6-chlorofebrifugine (now known as halofuginone), was claimed to have very marked anticoccidial activity (see Ryley & Betts, 1973). After a lapse of several years, this substance is now undergoing field development as an anticoccidial agent (Yvor6, Foure, Aycardi & Bennejean, 1974). In the present paper we present data on the anticoccidial activity of monensin, lasalocid and halofuginone under laboratory conditions, and describe some experiments carried out both in vivo and in vitro which throw light on their mode of action.
204
J. F. RYLEY AND B. G. WILSON
MATERIALS AND METHODS
Monensin has been obtained both in pure form and as a premix (Coban) by courtesy of Mr M. L. Clarke of Eli Lilly & Co. Lasalocid (Avatec) was kindly provided by Dr W. Rehm of Hoffmann-La Roche & Co. Ltd; most of the experiments were carried out with a 20 g sample lot No. 478-145 ARX, but the experiment summarized in Table 7 utilized material of lot No. 2936-94. This product is still under development, and standardization of purity and biological activity from batch to batch has not yet been achieved. Some of the halofuginone was supplied as 90% pure material by Dr E. Waletzky of American Cyanamid; other experiments were carried out using 'Stenoral' premix (Roussel-Uclaf), which contains 0-6% active ingredient in an inactive carrier. 'Lerbek' (Dow), a premix yielding at user level 100 ppm clopidol and 8-35 ppm methyl benzoquate, and robenidine (American Cyanamid) were used for comparative purposes. Biological methods used in the present study have already been fully described (Ryley & Wilson, 1971, 1972). Details of individual experiments are given in the tables, figures or text. Tables indicate days over which observations on mortality, weight gain and oocyst production were made. Day 0 in all cases was the day of inoculation; thus day — 1 represents the day before inoculation, day 3 represents 3 days after inoculation. Treatment 0—3 means that birds were fed medicated food from the time of inoculation until 72 h later, and thereafter unmedicated food; 2-12 means that birds received unmedicated food until 48 h after inoculation, and thereafter until day 12 diet containing drug. 0 -^ in Tables 1, 5 and 8 indicates that medicated food was supplied from the time of inoculation instead of 24 h previously as in the other groups. Blood in droppings was estimated visually on a scale of 0-3 for each cage; the faecal score is the average of all the replicates on the same treatment. RESULTS
Monensin Therapeutic activity Monensin is recommended for field use at an inclusion rate of 121 ppm. Table 1 summarizes cage experiments carried out with four species of coccidia using monensin at five different levels. In experiments of this type we have been in the habit of starting medication on the same day, but a few hours before, inoculation. With a variety of drugs (e.g. methyl benzoquate, robenidine, an azauracil derivative, etc.) we have been able to achieve complete coccidiosis control under such conditions. Not so with monensin. Preliminary experiments indicated that monensin displayed better activity when medication was started 24 h before inoculation, and slightly better still when given from 48 h before inoculation. Table 1 indicates that 121 ppm monensin given from 24 h before inoculation was unable completely to control an E. tenella challenge; some mortality and faecal blood were noted, weight gain was poor and there was a massive oocyst output. Control was even poorer when drug was administered from the time of inoculation only. Although increased incorporation rates of
Recent anticoccidials
205
Table 1. Anticoccidial activity of monensin in the chick E. tenella
E. necatrix
A
A
t
/o
Treatment (ppm drug in feed)
mortality (4-8)
Non-infected control Infected control Monensin 363 Monensin 242 Monensin 121 Monensin 60 Monensin 30 Monensin 121, 0-*
0-0 93-8 2-1 0-0 18-8 64-6 89-6 54-2
Av. wt. /o gain mor( - 1 to Oocysts Faecal tality (5-8) score (4-8) 8) — 0-0 0-9 80-5 — 21-6 2-7 70-8 00 0-0 34-3 0-08 63-8 0-0 2-28 0-0 00 62-3 15-76 1-7 2-7 12-5 28-1 — 21-3 16-7 2-8 43-2 4-78 2-0 250 E. brunetti
Av. wt. gain ( - 1 to Oocysts Faecal (5-8) score 8) — 81-0 00 26-3 30 31-8 0-00 0-0 63-4 0-00 0-0 841 10 0-50 1-07 2-5 551 0-90 3-0 511 0-70 62-3 2-8
E. acervtdina
A
A
1
/o
Treatment (ppm drug in feed)
mortality (4-7)
Non-infected control Infected control Monensin 363 Monensin 242 Monensin 121 Monensin 60 Monensin 30 Monensin 121, 0->
00 68-8 0-0 0-0 0-0 2-1 8-3 16-7
Av. wt. Av. wt. gain gain ( - 1 to Oocysts ( - 1 to Oocysts (4-7) (4-7) 7) V) . 66-0 67-8 3-2 — 12-5 1-76 36-4 301 017 0-90 55-6 461 1-30 0-85 52-4 64-3 7-51 4-71 35-9 47-8 6-81 6-84 29-5 6-04 230 7-61 370 41-8 609 5-87
Forty-eight birds per treatment; inoculated on day 0 with 403 000, 257 000, 250 000 or 4 x 106 oocysts of E. tenella, E. necatrix, E. brunetti and E. acervulina, respectively. Medicated food supplied from day — 1, except where indicated by 0-s-, which indicates medication supplied from time of inoculation only.
monensin resulted in better coccidial control, as indicated by reduced mortality, oocyst output and faecal score, weight gains were poor. Monensin apparently has an appetite-depressing effect, and 121 ppm seems to be a compromise between what the bird will take and what will give adequate anticoccidial activity. Rather better results were achieved with 121 ppm monensin against the other three species, though in all cases there was incomplete control as indicated by an appreciable oocyst output, and in each case better results were achieved when medication was started on the day before rather than the day of inoculation. Restricted medication Table 2 summarizes an experiment in which groups of 56 chicks were inoculated with 617000 oocysts of E. tenella and treated with food containing 242 ppm monensin over the periods indicated. This incorporation rate was chosen in an attempt to achieve maximum control when drug was present. It will be noted, however, that although control - at least as measured by mortality - was achieved
206
J. F. BYLEY AND K. G. WILSON
Table 2. Effect of restricted medication with monensin on chick mortality due to Eimeria tenella Days after inoculation Treatment , (days) 1 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-12 1-12 2-12 3-12 -1-12
— — — — — — — — — — — —
A
2
3
— — — — —
— —
—. — —
—. —
—.
.— .— — •
—
—
—
—
—
—
4
5
6
7
4 5 4 1 2 — — 1 3 2 4 10
33 13 8 3 2 4 5 7 3
8 9 9 6 1 3 — 3 1 1 8 4
3 6 1 — — 1
3 19 20
•
—
1 1 — — —
8
9
10
11
— — —. — — —. —.
— — — — — — —
1 3 1 1 — — 1 —
3 1
3 1 — — — —
. — — —
1
2 —
3
1 — —
, Total 12 deaths — 52 — 37 — 26 — 12 — 5 — 8 — 6 — 12 — 8 — 8 — 33 — 37 — 0
Fifty-six birds per treatment; inoculum, 617000 oocysts; monensin supplied at 242 ppm in diet.
when medication was given from day — 1 to day 12, in all groups where treatment started at the time of inoculation, some birds died from coccidiosis. With shorter periods of treatment, more birds died, but a clear mortality picture failed to emerge. Because E. brunetti seemed to be more susceptible (Table 1), a similar experiment was carried out using groups of 48 chicks inoculated with 190000 oocysts of this species and medicated for restricted periods with food containing 180 ppm monensin. Mortality was minimal, and so the parameters of daily weight and daily oocyst output were followed. Fig. 1 indicates that although continuous medication with monensin did not completely control weight gain, virtually an equivalent growth curve was obtained when drug was administered for the first 24 h of infection only. Likewise a delay in the start of medication of 1-4 days after inoculation improved growth somewhat compared to the infected unmedicated controls. All groups, however, showed some oocyst output, in every case reaching a maximum on days 6-7 (Table 3). Histological experiments Groups of 10-day-old chicks were fed diets containing 363, 242 or 121 ppm monensin and 3 days later were inoculated with 10 million oocysts of E. tenella or E. brunetti; control birds on unmedicated food were given 10 million, 200000 or 5000 oocysts. Two birds in each group were killed each day, and four pieces of caecum or five pieces of small intestine were taken, washed through with saline, fixed in Stieve's fluid (water, 570 ml; HgCl2, 34-5 g; 40% formaldehyde, 150 ml; glacial acetic acid, 30 ml), sectioned and stained with haematoxylin and eosin or PAS-AO. In this type of experiment, it was necessary to use very large inocula of oocysts in order to get enough first-generation stages to make microscopical observation
Recent anticoccidials 140
207
Non-infect. control 0-1 0-9
(A) 120
Non-infect. control 0-9
100 Infect, control
80
60 4 Days
4 Days
8
Fig. 1. Effect of restricted medication with monensin on growth of chicks infected with Eimeria brunetti. Forty-eight chicks per treatment, inoculated on day 0 with 190000 oocysts; diet containing 180 ppm monensin supplied over periods indicated.
Table 3. Oocyst output during restricted medication with monensin (same experiment as in Fig. 1 using Eimeria brunetti) Oocyst output (millions per chick) Treatment days
5-6
6-7
7-8
8-9
Total
Control 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-14 1-14 2-14 3-14 4-14 5-14 1-5
0-73 1-40 0-96 0-30 0-35 0-20 0-38 0-32 0-24 1-79 1-46 1-99 1-46 0-98 1-70
2-08 2-89 3-31 210 1-56 1-33 0-87 114 0-90 3-32 2-95 2-78 2-38 2-29 4-37
1-51 2-42 1-34 1-49 1-41 1-02 0-56 0-48 0-42 1-42 1-23 1-29 117 1-08 3-57
115 1-82 0-99 101 1-14 0-61 012 009 0-10 0-20 0-29 0-23 0-20 0-34 1-30
5-47 8-53 6-60 4-90 4-46 316 1-93 203 1-66 6-73 5-93 6-29 5-21 4-69 10-94
of the effects of drug on these stages possible. With E, tenella at the three drug levels tested, haemorrhage was noted in each case, but caecal core formation only at 121 ppm. At all three levels, all stages of the life-cycle could be observed microscopically at appropriate times, but there was an overall delay of up to 24 h in the development of each stage, and an overall appreciable reduction in the number of developing parasites when the size of the inoculum was taken into account. With 363 ppm monensin, some of the mature second-generation schizonts observed on days 4 or 5 contained abnormal rounded-off merozoites similar to those seen with lasalocid. In the case of individual parasites which failed to develop, inhibition appeared to be right at the start of the life-cycle; there was no accumulation of partially developed but inhibited parasites as has been observed with some drugs, e.g. robenidine (Ryley & Wilson, 1971). Similar
208
J. I\ EYLEY AND E. G. WILSON
effects were noted with E. brunetti; some parasites developed to produce sexual stages, but there was an appreciable reduction in the numbers so doing, particularly at 242 and 363 ppm. Tissue culture experiments Complete inhibition of E. tenella and E. brunetti in chick kidney tissue culture occurred with 0-002 ppm monensin. When 0-005 ppm drug was added to cultures inoculated with sporozoites of E. tenella, the parasites were inhibited as multinucleate first-generation schizonts. Vacuolation and degeneration were evident from 48 h, and little recognizable parasite material was found at 66 h. When cultures containing 0-005 ppm monensin were inoculated with sporozoites of E. brunetti, very few parasites developed beyond the trophozoite stage before undergoing degeneration; first-generation development of E. brunetti in culture is slower than E. tenella, and inhibition therefore set in at an earlier stage. No second-generation development was seen when cultures inoculated with E. tenella and containing 0-005 ppm monensin were washed free of drug 24, 48 or 72 h after inoculation. A few mature first-generation schizonts were seen subsequent to 24 h withdrawal, but these did not give rise to second-generation schizonts. When 0-005 ppm drug was added 24, 48 or 72 h after inoculation, inhibited parasites started to degenerate within 24 h of drug addition. When drug was added 96 h after inoculation, some second-generation schizonts matured, but the merozoites rounded off after release; late multinucleate second-generation schizonts became disrupted and vacuolated. When cultures were treated with 0-005 ppm drug for 24, 48 or 72 h periods starting 24 or 48 h after inoculation, no mature second-generation schizonts developed. However, treatment during the period 72-96 h allowed some schizonts to mature and release their merozoites apparently normally, while other merozoites became rounded and multinucleate schizonts vacuolated. When cell cultures were treated with 0-005 ppm monensin for 24 or 48 h and washed free of drug immediately before inoculation with E. tenella, no inhibitory effect was seen on either cell or parasite development. When, however, the preinoculation drug level was raised to 0-025 ppm (10 times the minimum active level) very few or no second-generation schizonts subsequently developed, although cell cultures appeared to be growing normally. Kidney cell cultures were grown for 24 or 48 h in medium containing 0-025 ppm drug and then allowed to continue growth in drug-free medium for periods up to 72 h before inoculation with sporozoites. Table 4 summarizes the effects on parasite development subsequently observed. Lasalocid Therapeutic activity Table 5 summarizes experiments carried out to determine dose-response relationships of lasalocid using three species of coccidia. Lasalocid at 75 ppm showed some activity under our experimental conditions, but did not give adequate control in the case of E. tenella or E. necatrix; with E. brunetti, although weight gain and mortality were controlled, there was a small oocyst output. In all cases,
Recent anticoccidials
209
Table 4. Effects of the preincubation of tissue culture cells with monensin on the subsequent development of Eimeria tenella Drug on
Drug off Inoculated
(h)
(h)
(h)
0 0
24 24
48 72
24
96
48 48 48
72 96 120
0 0 0
Parasite development Inhibited as multinucleate primary schizonts Inhibited as multinucleate primary schizonts; few mature schizonts produced, but no subsequent secondary development Some early secondary schizonts present at 168 h, but no mature forms developed subsequently Inhibited as multinucleate primary schizonts Inhibited as multinucleate primary schizonts Early secondary schizonts present at 192 h; no corresponding mature development seen at 216 h. Traces of mature secondary development at 240 h
150 ppm of the material used produced excellent results. Lasalocid at 75 ppm administered from the time of inoculation rather than 24 h previously produced approximately the same result; certainly there was not the marked falling-off in control observed with monensin. Lerbek and robenidine, at recommended levels, gave very good control with all species; monensin once again was rather disappointing, particularly with E. tenella. Restricted medication Table 6 summarizes an experiment carried out to indicate the stage in the life-cycle at which lasalocid acts. Although a level of 75 ppm is being recommended for field use (Mitrovic & Schildknecht, 1973, 1974), Table 5 indicates that at this level under our experimental conditions, incomplete control of the infections was achieved. Since 150 ppm of this batch of material did on the other hand give complete control, including elimination of oocyst production, this level was chosen for mode of action studies. As Table 6 indicates, 47/48 untreated birds died, the majority doing so on day 5. Treatment restricted to the first 24 h of the infection reduced mortality below 50%, and again the majority of birds which died did so on day 5; treatment for 2 or more days resulted in little mortality. Growth curves based on daily weighings (Fig. 2 A) show a similar picture; medication for the first day of the infection only gave partial control of the inhibitory effects of coccidia on growth, while medication for 2 or more days was adequate to give growth curves indistinguishable from the uninfected controls. Daily oocyst counts were also carried out on each group; Table 6 indicates total oocyst production over days 5-12 in each group expressed as millions of oocysts per chick. Medication for the first 4-5 days was sufficient to prevent almost completely oocyst production on drug withdrawal; no displacement of the day of maximum oocyst production was observed with shorter treatments. These experiments suggest that lasalocid exerts an initial coccidiostatic action at 150 ppm, but that with continued contact, this effect quickly becomes
Av. wt. /o gain mortality Oocysts (4-8) ( - 1 to 8) (5-8) . . 82-8 0-0 — 16-1 95-3 79-3 0-00 0-0 62-6 14-45 12-5 — 21-7 67-2 — 141 95-3 — 95-3 13-5 36-4 10-30 15-6 37-7 12-27 391 9-00 239 62-5 0-00 75-8 0-0 000 79-5 0-0 0-0 30 0-4 2-4 30 3-0 30 2-8 2-9 30 0-0 0-0
0-0 92-2 0-0 31 35-9 78-1 89-1 9-4 00 42-9 00 0-0 83-0 18-7 83-5 71-0 40-4 16-3 17-5 54-0 79-1 39-5 80-2 83-5 0-00 0-58 0-57 — — 0-47 0-65 0-41 0-02 0-00
Av. wt. /o Faecal mortality gain Oocysts score (4-8) ( - 1 to 8) (6-8)
A
0/
E. necatrix
A
^
E. tenella 0/
A
E. brunetti
0-0 2-8 0-0 0-1 1-5 1-8 2-6 1-3 01 2-1 00 0-0
1-30 000 0-00
67-7 19-4 71-8 71-9 690 47-8 32-7 70-6 70-6 70-3
0-0 20-3 0-0 00 00 0-0 6-3 1-6 .— 00 00
4-51 000 0-15 504 11-95 9-70
Av. wt. /o Faecal mortality Oocysts gain (4-7) (-Ito7) score (4-7)
i
Sixty-four birds per treatment; inoculated on day 0 with 646000, 222000 or 197000 oocysts of E. tenella, E. necatrix and E. brunetti, respectively. Medicated food supplied from day — 1, except where indicated by 0 ->•, which means medication supplied from time of inoculation only.
Non-infected control Infected control Lasalocid 150 Lasalocid 75 Lasalocid 37-5 Lasalocid 19 Lasalocid 9 Lasalocid 75, 0 -> Monensin 121 Monensin 121, 0 -> Lerbek 108 Robenidine 33
Treatment (ppm drug in feed)
t
Table 5. Anticoccidial activity of lasalocid in the chick
o
CO
w o
t)
H
w
o
Recent anticoccidials
211
Table 6. Effect of restricted medication with lasalocid on chick mortality due to Eimeria tenella Treatment days — 0-1 0-2 0-3 0-4 0-5 0-7 0-10 0-12 1-12 2-12 3-12
Days after inoculation , • —-*• 1 2 3 4 5 6 7 8 9 1 0 11 — — — 1 4 3 3 _ _ _ _ _ _ — — — — 14 3 2 1 1 — — _ _ _ _ _ i i _ _ _ _ _ _ _ _ _ _ _ _ i _ _ _ _ _ _ — _ — _ 1 — — — _ _ _ _ _ _ _ _ _ _ _ — — — — — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 1 — — — _ — _ _ _ _ 2 5 1 1 1 — — — _ _ _ 1 3 5 1 1 i _ _ _
Oocyst , Total production 12 deaths 5-12 47 — — 21 23-87 2 11-43 _ l 4-81 — 1 0-94 _ o 0-17 o 0-01 — o 0-00 o 0-00 — 3 0-75 — 28 1-31 _ 38 019
Forty-eight birds per t r e a t m e n t ; inoculum, 430000 oocysts; lasalocid supplied a t 150 p p m i n diet. 120
r
Non-infect. control
•
100
Non-infect. control
0-1
80
Infect, control
Infect, control
60 I
1
1
6 8 8 0 2 4 Days Days Fig. 2. Effect of restricted medication with lasalocid on growth of chicks infected with Eimeria tenella. Details as recorded in Table 6.
0
2
4
6
coccidiocidal; 2 day's treatment was sufficient to kill the majority of the coccidia as judged by mortality and weight gain control following drug withdrawal, while around 5 day's treatment was required to give a complete coccidiocidal effect as judged by oocyst production following drug withdrawal. The mortality pattern observed and the fact that medication had to start within 24 h of inoculation to prevent mortality (Table 6) suggests that lasalocid exerts its action somewhere during the first asexual cycle of reproduction. Table 7 summarizes an experiment carried out with E. tenella to examine the effects of lasalocid against later stages of the life-cycle. The first attempt to do this showed that 150 ppm of the new batch of lasalocid did not completely prevent oocyst production when given continuously from the time of inoculation; the table records daily oocyst counts carried out on groups of six chicks inoculated with 2000 oocysts on day 0 and given food medicated with 300 ppm lasalocid from the times indicated until the end of the experiment. It will be seen that if medication was started within 72 h of inoculation, no oocysts were produced; 14
P A R 70
212
J. F. RYLEY AND R. G. WILSON
Table 7. Effect of delayed treatment with lasalocid on the oocyst output of chickens infected with Eimeria tenella (Figures are oocyst outputs in millions/chick/day) Days postinfection
Period of treatment (days) Infected control
4-5 5-6 6-7 7-8 8-9
0-08 3-20 11-38 5-44
9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18
1-47 1-64 0-49 1-77 1-32 5-82 7-37 3-47 2-53
Total
50-09
411
A
0-18
1-18
2-18
3-18
4-18
5-18
6-18
7-18
000
0-00 0-00
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00
0-00 0-00 0-00
0-05 0-49 0-20
0-08 2-67
0-08 2-89 1111
000
009 001
3-26 1-16 0-16 0-02 0-01 0-00
0-06 1-93 7-71 5-25 2-30 0-89 0-37 0-04 0-05 0-00
0-00 0-00
000
000
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00
0-00
0-00
000 000
0-00 000 000
0-00 000
0-00 0-00 0-00
000 000
0-00 0-00 0-00
0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 0-00 000
0-00
0-00 0-00 000
0-00 0-00 0-00 0-00 0-00 0-00 0-84
101
000 000
814
2-91 0-47 0-22 0-36 0-03 0-01
000
000
000
0-00 0-07 0-02
8-37
18-69
0-00 0-00 0-00 26-22
0-00 0-00
when medication was started on day 4, few oocysts were subsequently produced, and even when medication was delayed until the infection had become patent, oocyst output was fairly quickly stopped. Histological experiments Chicks were inoculated with 10 oocysts of E. tenella and fed a diet containing 150 ppm lasalocid beginning at the time of inoculation or 24 or 48 h later. Two birds from each group were killed each day, and two pieces of each caecum taken for histology, being processed as before. When drug was present from the time of inoculation, both treated and control sections showed undeveloped intracellular parasites at 24 h. At 48 h, control sections contained appreciable numbers of medium and large multinucleate first-generation schizonts, and a few mature schizonts. Sections from treated birds showed far fewer parasites, and those that were present showed retarded development; some undeveloped sporozoites/ trophozoites were found, and a few small multinucleate and the occasional larger schizont were seen, mostly showing signs of vacuolation and degeneration. A few degenerating medium-sized first-generation schizonts were found at 72 h in treated birds, but no parasites could be found at 96 h; there was no evidence of tissue destruction or bleeding. By contrast, control birds showed massive bleeding and tissue destruction by 72 h (due to break-up of the first-generation schizonts produced from the very heavy inoculum) and countless developing second-generation schizonts, many of which were mature by 96 h. When medication was delayed until 24 h after inoculation, there was a retardation and reduction but not complete suppression of first-generation schizogony, and likewise an overall reduction in, but far from complete suppression of, second-
Recent anticoccidials
213
generation schizogony; bleeding and tissue destruction were evident as a consequence of second-generation schizogony, but many apparently mature schizonts contained rounded-off bodies rather than elongate merozoites. Subsequent experiments with a lower inoculum (5000 oocysts) indicated a very marked reduction in sexual development in birds where medication was started 24 h after inoculation. A 48 h delay in medication produced little evidence of drug activity at the histological level - in spite of the results recorded in Table 7 on eventual oocyst production. Tissue culture experiments No second-generation development was observed in cultures inoculated with E. tenella or E. brunetti and treated with 0-004 ppm lasalocid. When 0-01 ppm drug was added to cultures from the time of inoculation with E. tenella, some mature first-generation schizonts could be found from 48 h on. There were, however, fewer schizonts than in untreated cultures, and in cultures where merozoites were released and invaded fresh kidney cells, no further development was noted. With higher levels of drug, fewer schizonts matured; many schizonts were inhibited before or at merozoite formation. By contrast, addition of 0-01 ppm lasalocid to cultures along with sporozoites of E. brunetti resulted in inhibition of the majority of parasites at the sporozoite or trophozoite stage; a few small multinucleate first-generation schizonts together with the occasional mature schizont could be found at 64 h, but no further development was seen. Cultures containing 0-01 ppm lasalocid were inoculated with E. tenella, and the drug removed after various intervals. When drug was removed after 24 or 48 h, parasites were subsequently able to complete two asexual cycles of development, although the number of second-generation schizonts in the latter case was small; hardly any second-generation parasites were found when drug had been present for 72 h. Treatment with 0-01 ppm drug for a 24 h period during days 1-4 caused a delay in development, but many second-generation schizonts developed after drug withdrawal. Fewer second-generation schizonts developed when the cultures were treated for 48 h, and hardly any when the period of treatment was extended to 72 h. In cases where the limited treatment started 48 or 72 h after inoculation, some of the second-generation merozoites became rounded off before or after rupture of the schizont. No second-generation schizonts developed when 0-01 ppm drug was added 24 or 48 h after inoculation; first-generation schizonts matured and released their merozoites, but no further development was seen. When drug was added 72 or 96 h after inoculation, many normal second-generation schizonts developed, but others failed to produce normal merozoites, rounded-off bodies being released instead. No effect on parasite development was seen when cultures were pretreated with up to 0-1 ppm lasalocid (25 times the minimum effective level) for 48 h and then washed free of drug immediately before inoculation with sporozoites.
14-2
0-0 100-0 0-0 0-0 0-0 7-8 92-2 0-0 32-8 53-1 0-0 0-0
80-1 23-8 58-4 76-0 81-7 63-3 21-5 80-1 54-8 39-2 79-1 81-4 0-00 0-00 0-14 9-94 — 0-08 11-00 2-96 0-00 0-00
E. necalrix
00 2-1 0-0 0-0 0-0 1-3 2-5 00 2-5 2-5 0-0 00 0-0 92-2 0-0 0-0 5-2 500 921 8-1 111 60-7 0-0 00
66-4 9-6 52-8 62-3 59-6 25-8 10-8 65-4 53-0 30-7 67-9 63-9 . 0-00 0-00 0-01 0-11 — 0-05 0-52 0-66 0-00 0-00
Av. wt. /o Faecal mortality gain Oocysts score (4-8) ( - 1 to 8) (5-8)
t
A
0-0 30 00 0-0 0-3 2-5 2-9 0-4 1-6 1-9 0-0 0-0
0-0 31-3 0-0 0-0 00 10-9 10-9 0-0 0-0 1-6 0-0 0-0
66-8 13-6 47-9 60-9 57-8 29-8 19-5 561 58-6 43-4 60-7 61-8
Av. wt. /o gain Faecal mortality (4-7) ( - I t o 7 ) score 0/
E. bruneth
3-77 0-00 000 0-37 3-27 4-36 019 3-31 5-75 0-00 000
Oocysts (4-7)
Sixty-four birds per treatment; inoculated on day 0 with 319000, 250000 or 298000 oocysts of E. tenella, E. necatrix and E. brunetti, respectively. Medicated food supplied from day — 1, except where indicated by 0 ->, which means medication was supplied from the time of inoculation only.
Non-infected control Infected control Halofuginone 12 Halofuginone 6 Halofuginone 3 Halofuginone 1-5 Halofuginone 0-75 Halofuginone 3, 0 -*• Monensin 121 Monensin 121, 0 -> Lerbek 108 Robenidine 33
Treatment (ppm drug in feed)
0/
Av. wt. /o mortality Oocysts gain (5-8) (4-8) ( - 1 to 8)
A
E. tenella
Table 8. Anticoccidial activity of halofuginone. in the chick
O
m
P
S3
>
F
to
Recent anticoccidials
215 Non-infect. control 2-14
Non-infect. control 0-5 0-4 0-3
140 120
3-14
0-2
100
0-1 80
Infect, control
Infect, control
60 I
4 Days
4 Days
Fig. 3. Effect of restricted medication with halofuginone on growth of chicks infected with Eimeria tenella. Details as recorded in Table 9.
Halofuginone Therapeutic activity Halofuginone has been evaluated at five inclusion rates against three species of coccidia (Table 8). Complete control of mortality and oocyst production was achieved with 6 ppm, and quite good control with 3 ppm. It should be noted that control dropped markedly when the inclusion rate was reduced below 3 ppm, and that weight gains were not as good as the controls at 6 ppm and markedly reduced at 12 ppm, indicating toxicity. Once again, excellent control of all species was achieved with Lerbek and robenidine, and only partial control by monensin. Unlike the situation with monensin, medication with halofuginone could be started at the time of inoculation and still produce satisfactory results. Restricted medication
Therapeutic experiments (Table 8) indicated that 3 ppm just about gave control of the coccidial challenges used, but in view of the rather steep doseresponse curve with halofuginone, levels of 4-5 and 6 ppm have been used in some of the mode of action studies hopefully to obtain more decisive effects. Table 9 and Fig. 3 summarize an experiment carried out using 4-5 ppm halofuginone in restricted schedules of medication. Oocyst production data (Table 9) indicates that in this particular experiment, complete suppression was not achieved by continuous medication, although no birds died and there was little evidence of infection from growth data. That the drug was not immediately coccidiocidal under these conditions is apparent from the relapses indicated by mortality and oocyst production following drug withdrawal after administration for 1-5 days; growth curves gave a rather less sensitive indication of relapse. To avoid mortality, drug treatment had to be started within 24 h of infection, although a delay of 48 h resulted in but few deaths and a growth curve indistinguishable from that produced under continuous medication. An experiment was carried out to investigate the effects of halofuginone at 6 ppm against the later stages of the life-cycle of E. tenella concurrently with
0-4 0-5 0-7 0-10 0-14 1-14 2-14
Treatment days 8 19 7 3 — 1 — 7 6 1 .— 1
— — 13 2 — —
•— 1 2 9 .— —
— — 2 12 10 — ,
10 — — — — 7 2 ,
11 — .— .— .— 1 5 .
12 — — — — — 4 1
13
14
Sixty-four birds per treatment; inoculum, 409000 oocysts; halofuginone supplied at 4-5 ppm in diet.
2 26
54 24 1 1 — —
Days after inoculation
63 51 31 28 18 13 2 0 0 0 3 32
— 43-20 45-24 53-05 35-20 3-57 2-27 2-29 215 2-66 6-06
Oocyst Total production 5-14 deaths
Table 9. Effect of restricted medication with halofuginone on chick mortality due to Eimeria tenella
to
CF O Q
M
?
o w
>
M
K|
OS
Recent anticoccidials
217
Table 10. Effect of delayed treatment with halofuginone on the oocyst output of chickens infected with Eimeria tenella (Figures are ooeyst outputs in millions/chick/day) Days postinfection 4-5 5-6 6-7 7-8 8-9
9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 17-18 Total
Period of treatment (days) Infected control 0-09 2-55 11-48 5-90 4-34 2-96 2-43 1-79 0-53 0-66 4-25 8-83 8-91 3-80 58-52
A
, 0-18
1-18
2-18
3-18
4-18
5-18
6-18
7-18
0-00
0-00 0-00 0-00 0-01
000
0-00 0-00 0-02 0-03
0-05 0-49
0-07 0-95 0-41 1-34 0-19
0-08
0-08 2-67 12-09 8-68 6-77 2-29 0-67 0-20 0-01 0-01
000
0-00 0-00 001 000 000
0-00 000 000
0-00 0-00 0-00 0-00 001
000
0-00 0-00 0-00 0-00 000
0-00 0-00 0-00 0-00 0-01
0-00 0-08 0-11 0-02 0-00 0-00 000 000
000
0-00 0-00 0-02 0-00 0-00 0-00 0-00
0-00 0-00 0-00 0-00 0-00
000 000
0-21
0-07
006
0-06 0-00 0-00 0-00 000
0-00 000 000
0-00 0-00 0-00 0-66
003
0-00 0-00 000 000
0-00 0-00 000
0-00 2-99
216
8-25 5-30 1-66 0-23 0-04 000 000
0-00 0-00 0-00 0-00 0-00 17-72
003
0-00 000
0-00 33-50
that recorded for lasalocid in Table 6. As Table 10 indicates, oocyst outputs were negligible when medication was started at any time up to 96 h after inoculation; medication started once the infection had become patent resulted in a fairly rapid cessation of oocyst production and a reduction iri total output compared with the control. Histological experiments Chickens inoculated with 107 oocysts of E. tenella were treated in groups with diet containing 6 ppm halofuginone starting at the time of inoculation or 24 or 48 h later. Two birds from each group were killed each day and two pieces of tissue taken from each caecum for histology. Examination of slides stained with haematoxylin and eosin or PAS-AO indicated that when halofuginone was present from the time of inoculation, no coccidial development took place; parasites invaded crypt epithelial cells, a parasitophorous vacuole was formed, but the parasites rounded off, degenerated, and were quickly eliminated. When medication started 24 h after inoculation, some small and medium-sized first-generation schizonts were seen at 48 h, some of which appeared to be vacuolated and degenerating. At 72 h there were some vestigial remains of degenerating first-generation parasites, but no trace of second-generation parasites; sections were clear of coccidia by 96 h. When drug administration was delayed until 48 h after inoculation, no consistent anticoccidial effect could be discerned at the histological level. In a repeat experiment involving 3 ppm drug, most of the parasites failed to develop at all when drug was present from the time of inoculation, although a few underwent the complete life-cycle with a 24 h delay at each stage. Following a 24 h delay in the start of medication with 3 ppm drug, plenty of parasites could
218
J. F. RYLEY AND R. G. WILSON
be found at each stage, though there was obviously an overall reduction in numbers in view of the immense inoculum. No effect could be noted histologically when drug treatment was started 2 or 3 days after inoculation - although the experiment recorded in Table 10 indicates the ability of late treatment to suppress oocyst production. Tissue culture experiments The drug showed complete activity against E. tenella at 0-01 ppm. Incomplete and variable activity was achieved with 0-005 or 0-002 ppm, and no activity with 0-001 ppm. Continuous treatment with 0-01 ppm drug prevented any development beyond the sporozoite stage. Similar levels of drug showed similar effects with E. brunetti. When drug (0-01 ppm) was removed 24 h after inoculation, numerous mature second-generation schizonts could be found 5 days later; fewer parasites developed following drug withdrawal at 48 h, and none when drug was withdrawn at 72 h. When cultures were treated with 0-01 ppm drug for 24 h starting 24, 48 or 72 h after inoculation, all produced mature second-generation schizonts 3-5 days after drug withdrawal. If cultures were treated for 48 h, starting 24 or 48 h after inoculation, only a limited amount of second-generation schizogony was produced after drug withdrawal. When cultures were treated over the period 24-96 h after inoculation, only a few multinucleate first-generation schizonts had developed 3 days after withdrawal, and no second-generation development took place. Starting treatment with 0-01 ppm drug 24, 48, 72 or 96 h after inoculation showed that the drug was active against life-cycle stages other than the sporozoite. Administration commencing 24 h after inoculation resulted in inhibition at the sporozoite or trophozoite stage. Following addition at 48 h, immature firstgeneration schizonts failed to mature, but became vacuolated and degenerate; mature first-generation schizonts present at the time of drug addition failed to develop further. When drug was added at 72 h, early second-generation schizonts became vacuolated and degenerate. Similar effects were produced when drug treatment was started at 96 h, although mature forms present produced merozoites and subsequently released them. Development of drug resistance
Eight passages of E. tenella have been carried out in groups of three, 3-week-old chicks reared in plastic-film isolators, in the presence of increasing levels of halofuginone. In the initial passage, which involved an inoculum of 106 oocysts and a drug level of 3 ppm, faecal blood and caecal cores were produced. The total oocyst yield from this passage was inoculated into further chicks given food containing 6 ppm halofuginone; blood and caecal cores were again produced. Five further passages have been achieved in the presence of 12 ppm halofuginone followed by one with 18 ppm. Moderate numbers of oocysts were obtained at each passage, but no blood or caecal cores were noticed. The coccidia developing in the presence of 18 ppm halofuginone appear to be E. acervulina, presumably derived from an unsuspected slight contaminant in the original culture!
Recent anticoccidials
219
DISCUSSION
Under the experimental conditions reported here, Lerbek and robenidine gave very good control of the three species of coccidia tested. Over the past 30 years there has been a succession of anticoccidial agents developed, each giving better control under experimental conditions, often at a lower absolute dose, than its predecessors. It is hard to imagine how a new drug could be launched which did not appear better than currently available anticoccidials. Although few details of its anticoccidial activity have been published (see Ryley & Betts, 1973), monensin, currently the most commonly used anticoccidial agent in the U.S.A. is not very impressive by comparison with the other drugs in stringent laboratory tests. In the present studies it compares very unfavourably with Lerbek, robenidine, lasalocid or halofuginone, yet it is undoubtedly very successful commercially. This fact causes concern to the experimental coccidiologist who wonders what criteria of activity in the laboratory to apply to a compound in order to decide to take it forward to field trial. Under experimental conditions, monensin seems to require time to build up tissue levels adequate to show maximum activity. Although this of course is of no consequence under field conditions as a prophylactic agent, it does make it difficult to devise satisfactory experimental studies on its point of action. At an incorporation rate of 121 ppm - a compromise between activity and toxicity and/or appetite depression - it does not prevent development of all the parasites in a coccidial challenge. One must, therefore, assume that under field conditions severe challenges are seldom met, and that the drug is able to deal with the actual challenge sufficiently to prevent clinical coccidiosis. If some coccidial development does take place in the presence of drug, presumably adequate immunity develops to prevent later breakdown as the coccidial population in the environment increases (see Reid, Kowalski & Rice, 1972). Our experimental rations are deficient in vitamin K; although a commercial ration containing adequate vitamin K will enable monensin to give better control of the haemorrhagic species in the field, it will not alter the relative efficacies of the various drugs. Jeffers (1974) has published a fascinating study of 308 strains of E. tenella isolated from 1145 litter samples obtained from all the major broiler-producing regions in the USA. Using a somewhat crude method of evaluation, he found, among other things, that the majority of strains isolated from enterprises using 'efficient' anticoccidial drugs, such as Amprol Plus, clopidol, buquinolate or decoquinate, showed resistance to the particular drug being used. Although enterprises using monensin yielded an above average number of isolates of E. tenella (37-8%), none of these showed a reduced sensitivity to the drug. It should be remembered that monensin has not been in use for as long a period of time as the other drugs where resistance has emerged. It is possible that organisms find it difficult to develop resistance against compounds such as monensin, which are interfering with ion transport through membranes, rather than an enzymic reaction which could be by-passed. The possibility also exists that monensin does not act directly on the parasite at all, but rather modifies
220
J. F. RYLEY AND R. G. WILSON
the host cell in some way, making the environment unsuitable for parasitization — another situation in which it would be difficult to visualize the development of drug-resistance. In vitro experiments in which cultures were incubated with monensin and then washed free from drug prior to inoculation with parasites suggest, however, that indirect activity through the host cell probably does not occur. The fact that high concentrations of monensin have to be used to produce antiparasitic effects suggests that drug is absorbed and bound by the cell in a form, which although not removed by medium changing, is able to inhibit parasite development. Dilution of bound drug consequent on repeated cell division would explain the decreasing effects found after increasing intervals between drug removal and inoculation. I t could well be that under field conditions where cycling of coccidia is taking place in the presence of monensin, an inherent limitation in resistance development coupled with an effectively low drug pressure prevents the emergence of drug-resistant lines. Lasalocid is an ionophore bearing some relationship to monensin. In the present studies we were able to achieve complete control of three species of coccidia with 150 ppm drug. Insufficient material was available to determine the maximum tolerated level, but the manufacturer indicates that this depends on purity, which varies from batch to batch. It is apparent, however, that there is a greater separation of activity and toxicity than is the case with monensin, and it will be most interesting in the light of the above discussion to see whether lasalocid, superior to monensin in these laboratory experiments, is more or less successful than monensin under field conditions. Halofuginone is weight for weight one of the most active anticoccidials known. In our experience it shows a steeper dose-response curve than most, but the separation of activity from toxicity is minimal; our observations are in general similar to those recently reported by Yvore et al. (1974). It will be interesting to see how it behaves under field conditions, since the achievement of uniform incorporation rates at such a low level is difficult, and there is so little room for manoeuvre in the acceptable level. As a potentially 'efficient' anticoccidial, and in view of the limited laboratory work on resistance reported, it will be most interesting to note the speed of development of drug resistance under field conditions. The experiments described with monensin illustrate the difficulties which may be encountered when trying to pin-point the stage of the life-cycle at which an anticoccidial drug acts. Restricted medication experiments drew attention to the speed of action of the drug once adequate concentrations had been built up in the host, but did not indicate conclusively the point of action; in birds which managed to survive, very short periods of treatment were as effective as continuous medication. Likewise histological studies were difficult to interpret as a proportion of the parasites were able to complete the life-cycle. Nevertheless, the impression was gained that if an individual parasite was going to be inhibited, it would be inhibited right at the start of the parasitic phase. In vitro experiments enabled studies to be carried out under conditions where complete inhibition, uncomplicated by host toxicity, was possible. Such investigations underlined the
Recent anticoccidials
221
potentially rapid lethal action of monensin, but with E. tenella, drug action was sufficiently slow to allow some first-generation nuclear division to occur. With lasalocid it was necessary to use drug concentrations double that which may be recommended for field use in order to achieve complete interruption of the life-cycle. With 150 ppm drug, it was possible to demonstrate a fairly rapid lethal action on the parasite, and to indicate that under normal conditions of continuous administration, inhibition would occur very early in the endogenous cycle. Histological experiments showed that some parasites could undergo partial development, but few progressed further than halfway through the first asexual phase; slightly more development was possible in vitro, but in neither case did any second generation development occur. In vitro and in vivo experiments indicated that some relapse of development was possible following very restricted periods of treatment, but that in general all stages of the parasite were susceptible to lasalocid, although possibly not quite as sensitive as the very first intracellular forms. It must be emphasized that interesting though the sensitivity of later stages to a drug may be, the only observations of real significance are what happens when the drug is there from the start; in the field drug is continuously administered, and - failing a breakdown in the feeding regimen for any reason — is present when a coccidial challenge occurs. With halofuginone, possibly elevated drug levels had likewise to be used effectively to define the point of drug action. Once again it was the very earliest intracellular stage which was susceptible, but the drug appeared to be somewhat slower in producing a killing effect in that relapses could occur if medication was withdrawn within 5 days. In the experiments reported, attempts have been made to obtain more useful information from restricted medication experiments by daily weighings of birds and daily oocyst counts in addition to the mortality pattern previously used alone (Ryley, 1967; Ryley & Wilson, 1971). The additional information in fact obtained was of limited value, although we have found that with drugs having a pronounced coccidiostatic rather than coccidiocidal effect, e.g. robenidine, such observations can be very informative. We are grateful to Mrs Judith Hazlehurst, Mrs Thelma Robinson and Miss Linda Hardman for invaluable technical assistance.
REFERENCES
T. K. (1974). Eimeria tenella: Incidence, distribution, and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Diseases 18, 74-84. MITBOVIC, M. & SCHILDKNECHT, E. G. (1973). Anticoccidial activity of antibiotic X-537A in chickens. Poultry Science 52, 2065. MITBOVIC, M. & SCHILDKNECHT, E. G. (1974). Anticoccidial activity of Lasolocid (X-537A) in chicks. Poultry Science 53, 1448-55. REID, W. M., KOWALSKI, L. & BICE, J. (1972). Anticoccidial activity of monensin in floorpen experiments. Poultry Science 51, 139-46. RYLEY, J. F. (1967). Studies on the mode of action of quinolone and pyridone coccidiostats. JEFFEBS,
Journal of Parasitology 53, 1151-60.
222
J. F. RYLEY AND R. G. WILSON
RYLEY, J. F. & BETTS, M. J. (1973). Chemotherapy of chicken coccidiosis. In Advances in Pharmacology and Chemotherapy, vol. xi (ed. S. Garattini, A. Goldin, F. Hawking and I. J. Kopin), pp. 221-93. New York, London: Academic Press. RYLEY, J. F. & WILSON, R. G. (1971). Studies on the mode of action of the coccidiostat robenidene. Zeitschrift fur Parasitenkunde 37, 85-93. RYLEY, J . F . & WILSON, R. G. (1972). Comparative studies with anticoccidials and three species of chicken coccidia in vivo and in vitro. Journal of Parasitology 58, 664-8. YVORE, P., FOUBE, N., AYCARDI, J. & BENNEJEAN, G. (1974). Efficacite du Stenorol
(RU 19110) dans la chimioprophylaxie des coccidioses aviaires. Recueil de Medecine Veterinaire 150, 495-503.
Printed in Great Britain
