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Relative virulences of a drug-resistant and a drug-sensitive strain of Eimeria acervulina, a coccidium of chickens
Veterinary Parasitology 135 (2006) 15–23 www.elsevier.com/locate/vetpar
Relative virulences of a drug-resistant and a drug-sensitive strain of Eimeria acervulina, a coccidium of chickens R.B. Williams Veterinary Research Division, May & Baker Ltd., Ongar, Essex, UK Received 20 August 2005; received in revised form 3 November 2005; accepted 3 November 2005
Abstract The virulence of a field strain of the chicken coccidian parasite Eimeria acervulina (Boreham I), dually resistant to the chemically unrelated anticoccidial agents decoquinate and clopidol, was compared with that of a drug-sensitive laboratory strain (Ongar) of the same species. Following a single heavy infection (prevented from recycling), both strains exhibited pathogenic effects typical of their species, viz., pathognomonic lesions, adverse effects on body weight gain and feed conversion ratio (FCR), but no mortality. One week after infection, chicks infected with either strain had a statistically significantly worse weight gain than the uninfected control; the Boreham I strain produced more oocysts, and caused slightly more severe duodenal lesions and poorer FCRs than the Ongar strain (all those effects being non-significant). After 3 weeks, there were no significant differences between any cumulative effects of either strain, nor any differences from the uninfected control. However, from 2 to 3 weeks after infection, chicks infected with either strain had a greater feed consumption and growth rate than uninfected chicks. When chicks reared on solid floors were given lighter infections of either strain, which were allowed subsequently to recycle naturally, there were no consistent reductions in weight gains, but feed consumption was higher than that of uninfected chicks. Whatever, the mode of infection, there were no significant differences between the weights of infected and uninfected chicks after 3 weeks, but the FCR of infected chicks was usually poorer than that of uninfected chicks. The difference between the virulences of the Boreham I and Ongar strains was not greater than that between various drug-resistant strains or between various sensitive strains of several Eimeria species recorded in the literature. It is therefore concluded that there was no difference between the virulences of the two strains of E. acervulina that could be attributed to the drug-resistance of one of them. # 2005 Elsevier B.V. All rights reserved. Keywords: Chickens; Coccidiosis; Drug-resistance; Eimeria acervulina; Pathogenicity; Virulence
1. Introduction Despite drug-resistance in poultry coccidia being so well documented, it is still virtually impossible to predict what, in general, is its effect on commercial E-mail address: [email protected].
flocks. Most statements regarding the development of resistance to commercially available drugs in the field are general, anecdotal and second-hand, and are not based on experimental studies (e.g., McManus et al., 1968; Ryley, 1980; McDougald, 1982, 1990). The few records of particular resistance-related coccidiosis outbreaks in the field (e.g., Millard,
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.11.004
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1970; Chapman, 1982) include no detail that might help to elucidate the relevant circumstances. However, a unique study on the emergence of resistance of coccidia to decoquinate in some of the first broiler flocks that received the drug provided detailed information on coccidial population dynamics, drug-sensitivity testing of isolates of several Eimeria species, and the commercial performances of the affected broiler flocks (Williams, 2006). Although decoquinate-resistance of E. acervulina, E. brunetti, E. maxima and E. tenella developed during rearing of the first flocks to be fed the drug on some UK farms, no adverse effect on their performances was apparent (Williams, 2006). Several factors probably contributed to this unexpected outcome, including initially low levels of exposure to the more pathogenic drug-resistant species when each flock was placed on clean litter, and the stimulation of host immunity by continuous exposure to parasites that survived drug action. A further possibility is that the coccidia that had developed drug-resistance might have become concomitantly less virulent. Because very little has been published on any possible relationship between drugresistance and virulence of coccidia, that hypothesis has now been tested. The virulence of a strain of decoquinate-resistant E. acervulina isolated from one of the flocks monitored by Williams (2006) has been compared with that of a drug-sensitive laboratory strain. The results are assessed in the context of the sparse relevant literature.
2. Materials and methods 2.1. General This work was carried out in the same laboratory, using the same methods and facilities as those documented by Williams (1973). One-day-old male hybrid chicks (Rhode Island Red males Plymouth Rock females) were maintained free from coccidial infection until used at 1-week-old. During experiments the birds were reared in groups of five, in wire mesh-floored or solid-floored cages under continuous lighting in heated rooms, and allowed free access to water and a diet as described by Ball and Warren (1965). Water and feed contained no medication.
The Boreham I strain (B) of E. acervulina was isolated in this laboratory in May 1968 by the method of Williams (1969) from a commercial broiler house during the study reported by Williams (2006). It expressed dual resistance to decoquinate (‘Deccox’ – May & Baker Ltd.) and clopidol (‘Coyden 25’ – Dow Chemical Co.) (Williams, 1998). The laboratory strain of E. acervulina used as a control was the Ongar strain (O) described by Ball (1966), which was isolated in this laboratory in February 1958, well before decoquinate and clopidol became commercially available. It was sensitive to a wide range of contemporarily used anticoccidial drugs (Ball, 1966), including decoquinate (Hodgson, 1968). Infections were administered to chicks as aqueous suspensions of sporulated oocysts, by inoculation directly into the crop with a glass pipette. The oocysts of both strains tested were of the same age, and had been harvested from donor birds ca. 3 weeks before use. Oocysts produced in the faeces of chicks were counted by a modification of the method of Long and Rowell (1958), described by Williams (1973). 2.2. Experimental designs 2.2.1. Single infections with E. acervulina Thirty individually identified chicks per treatment were accommodated in six wire mesh-floored cages. Chicks in treatment 1 were uninfected; chicks in treatment 2 were each infected with 500,000 sporulated oocysts of E. acervulina (B); and chicks in treatment 3 were similarly infected with E. acervulina (O). Such a heavy infection is a ‘‘crowded dose’’ as defined by Williams (2001), and was necessary to produce a significant reduction of chick body weight gain. Twenty individually identified chicks in each of the three treatments were weighed singly 1 day before infection and thereafter weekly for 3 weeks. The remaining 10 chicks per treatment were killed on the fifth (six birds) and the sixth day (four birds) after infection, and the pathognomonic lesions in the duodenum and jejunum were scored as , +, ++ or +++ (negative, slight, moderate or severe). Faeces for oocyst counts were collected daily from under each of the four cages of chicks maintained for weighing, from the third day after infection until no oocysts were detected for two consecutive days. The oocysts produced by the birds in each cage were totalled,
R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
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from which the mean oocyst production per bird was calculated for each strain. The reproductive potential (RP) of each strain was also calculated (the number of oocysts produced by each bird per oocyst administered). Daily feed consumption by the chicks being weighed was measured throughout on a cage basis (four per treatment), and cumulative feed conversion ratios (FCRs) were calculated at the end of each week. FCR = cumulative total of feed eaten (kg)/cumulative total weight gain (kg) of birds in a cage. 2.2.2. Recycling infections with E. acervulina The object of this design was to assess the effect of allowing experimentally infected chicks to reinfect themselves repeatedly by facilitating access to their own faeces, as occurs naturally in the field. A sheet of corrugated cardboard completely covered the wire mesh floor of each cage, so that autoreinfection of infected chicks might occur. Ten individually identified chicks per treatment (in two cages) were used. Chicks in treatment 1 were uninfected; chicks in treatment 2 were each infected with 5000 sporulated oocysts of E. acervulina (B); and chicks in treatment 3 were similarly infected with E. acervulina (O), the doses being intended to mimic a moderate field challenge. Five fresh individual faeces per cage were collected and examined using the method of Hodgson (1970) after 1, 2 and 3 weeks to confirm that recycling had occurred in the infected chicks and that uninfected chicks remained free from coccidia. All chicks were individually weighed 1 day before infection and thereafter weekly for 3 weeks. Daily feed consumption was measured throughout on a cage basis (two per treatment), and cumulative FCRs were calculated for each cage at the end of each week. 2.3. Statistical analyses Methods and results of statistical analyses are given with the experimental results where appropriate.
Fig. 1. The mean daily oocyst production of the Boreham I and Ongar strains of E. acervulina in unmedicated chicks inoculated with 500,000 sporulated oocysts each.
period comprised days 4–13 after infection. The mean total oocyst production per bird for strain B was 116 million 9.8 million S.E. (RP = 232), and that for strain O was 83.5 million 8.5 million S.E. (RP = 167). The difference was not statistically significant by a t-test (two-tailed, P > 0.05). The uninfected controls excreted no oocysts. The mean weight gains of infected and uninfected chicks at weekly intervals during 3 weeks are shown in Table 1. Both strains caused a statistically significant weight gain depression during the first week (twotailed t-test, P < 0.05), but they were not significantly different from each other. From the end of the first week to 3 weeks, there were no significant differences between the weight gains of birds in the infected treatments or between birds in each of them and the uninfected treatment. No birds died. Fig. 2 shows the mean amount of feed eaten per infected chick, expressed relatively to that eaten by uninfected chicks during 3 weeks. The feed intake of infected chicks was reduced until the ninth day after infection but it subsequently exceeded that of Table 1 Weight gains (g S.E.) of chicks in wire mesh floored cages after a single infection of 500,000 sporulated oocysts each of E. acervulina
3. Results
Strain
After 1 week
After 2 weeks
After 3 weeks
3.1. Single infections with E. acervulina
Uninfected Boreham I Ongar
59.1 1.97 50.9 3.56a 50.1 3.56a
145.6 4.82 127.9 7.62 131.3 8.16
230.0 8.05 229.8 10.05 238.9 12.06
Fig. 1 shows the mean daily oocyst production per chick of the two strains of E. acervulina. The patent
a Each statistically significantly less than the controls (P < 0.05), but not significantly different from each other.
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23 Table 4 Weight gains (g S.E.) of chicks with a recycling infection of 5000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
73.1 2.97 75.7 2.42 71.1 2.59
173.0 6.55a 167.7 5.53 152.0 5.71a
278.5 10.96 278.8 8.98 276.5 9.50
a Just statistically significantly different from each other (P = 0.05).
between FCRs of infected and uninfected chicks were much smaller and the FCR of birds infected with strain O was better (lower) than that of uninfected controls. The magnitudes of differences between strain B and strain O after 1, 2 and 3 weeks were somewhat inconsistent. The observed differences were not amenable to valid statistical analysis because of the wide variation within the small numbers of replicate cages. Pathognomonic duodenal lesions, comprising white plaques, occurred in chicks killed 5 or 6 days after infection (Table 3). Strain B caused more severe lesions than strain O, but the difference (after the necessary pooling of cells in the contingency table to construct a 2 2 table) was not statistically significant (Fisher’s Exact test, P > 0.05).
Fig. 2. The mean feed consumption of chicks (g per day) infected with 500,000 sporulated oocysts each of the Boreham I or Ongar strain of E. acervulina, shown relatively to that of uninfected chicks.
Table 2 FCRs of chicks in wire mesh floored cages after a single infection of 500,000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
1.95 2.11 2.18
2.16 2.38 2.36
2.65 2.74 2.59
3.2. Recycling infections with E. acervulina Monitoring of fresh individual droppings for oocysts confirmed that the chicks infected with either strain continued to recycle infections throughout the experiment, and that the control chicks remained uninfected. Table 4 shows the mean weight gains of infected and uninfected chicks at weekly intervals during 3 weeks. Apart from the result with strain O after 2 weeks, which was only just statistically significant, there were no other differences between the weight gains of any of the treatments. No birds died.
uninfected chicks. Before the ninth day, chicks infected with strain O ate more than those infected with strain B. After the eighth day, the reverse was true. The last day of oocyst production was day 13. The mean FCRs of chicks at weekly intervals during 3 weeks are shown in Table 2. Chicks infected with either strain of E. acervulina had markedly poorer (higher) FCRs at 1 and 2 weeks after infection than uninfected chicks. After 3 weeks, differences
Table 3 Numbers of chicks with coccidial lesions of different severities, 5 or 6 days after a single infection of 500,000 sporulated oocysts each of E. acervulina Strain
Uninfected Boreham I Ongar
Severity of lesions on day 5 after infection
6 1 4
Severity of lesions on day 6 after infection
+
++
+++
0 2 2
0 1 0
0 2 0
4 2 1
+
++
+++
0 1 3
0 1 0
0 0 0
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4. Discussion 4.1. Virulence of coccidia
Fig. 3. The mean feed consumption of chicks (g per day) infected with 5000 sporulated oocysts each of the Boreham I or Ongar strain of E. acervulina, subsequently allowed to recycle, shown relatively to that of uninfected chicks.
Overall, infected chicks ate more than uninfected chicks (Fig. 3). However, there was an inexplicably reduced feed consumption relatively to uninfected controls during days 14–16 by birds infected with strain O. Nevertheless, chicks infected with strain B consistently ate more than those infected with strain O. Table 5 shows the mean FCRs at weekly intervals during 3 weeks. As expected from Fig. 3, infected chicks had poorer FCRs than uninfected chicks. As in the previous experiment, after 3 weeks the FCR of chicks infected with strain B was poorer than that of chicks infected with strain O. Differences between treatments were somewhat inconsistent during each time interval and were not amenable to valid statistical analysis because of the wide variation within the small numbers of replicate cages.
Table 5 FCRs of chicks in solid-floored cages, with a recycling infection of 5000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
2.10 2.24 2.17
2.59 3.13 3.24
2.93 3.25 3.09
To establish whether expression of drug-resistance might be linked with the capacities of the strains of E. acervulina studied to cause disease, it is first necessary to clarify some terminology. The observed effects of coccidia on chicks may be dealt with under two headings, viz., pathogenesis (the characteristic capacity of an organism to cause disease, and its course of development), and virulence (the disease-producing power, or degree of pathogenicity of an infection). Hence, in the present context, pathogenicity may be regarded as a qualitative property of a species, and virulence as an intraspecific quantitative effect. Whilst the pathogenic effects of coccidia are fairly well understood, the basis of virulence is not so easily established. The virulence of any particular strain of an Eimeria species might be considered to comprise two broad elements, one that may be manipulated and one that is inherent. The difficulty lies in separating the two. It is well known that administering increasingly higher numbers of sporulated oocysts to birds leads to greater virulence (Dickinson, 1941; Gardiner, 1955). The relationship between numbers of oocysts administered and the resulting progeny of oocysts excreted is also well established, and involves a crowding effect at higher oocyst doses (Williams, 1973, 2001). One might, therefore, intuitively expect that a defined oocyst inoculum, the resulting oocyst production and the severity of the pathogenic effects would be interlinked in some predictable way. However, this is not necessarily the case, because of differences between the inherent virulences of coccidial strains. The results of the present study must be considered in that context. 4.2. Pathogenesis of E. acervulina The pathogenic effects of the Boreham I and Ongar strains conformed in general to those usually recognised for E. acervulina (e.g., Joyner and Long, 1974; Long et al., 1976). Thus, there was no mortality, but pathognomonic lesions comprising white plaques were evident in the duodenum and jejunum, with adverse effects on bird weight gain and FCR. Weight gain reduction occurred during the week following the single heavy infection, after which there
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was a compensatory increase in growth rate. This increase was so marked that 3 weeks after infection, infected chicks were as heavy as or heavier than the uninfected controls (Table 1). Such a phenomenon in chicks receiving single infections was not observed by previous workers who terminated their experiments after only 2 weeks (Hein, 1968; Preston-Mafham and Sykes, 1970). Neither did Reid and Johnson (1970) and Michael and Hodges (1971) record complete compensation for weight gain reductions by 4 weeks in chicks infected once. In the case of recycling light infections in the present study, there were no consistent adverse effects on bird weight gain up to 3 weeks after the initial infection (Table 4). Although infected chicks were here able to compensate for weight gain reduction due to heavy E. acervulina infections or to maintain a normal growth rate in the presence of a light recycling infection, they ate more feed during recovery from the single heavy infection (Fig. 2) and during the whole course of the recycling light infection (Fig. 3) than uninfected chicks. Michael and Hodges (1971) also found that chicks recovering from a heavy infection had an increased feed consumption. Reid and Pitois (1965) demonstrated a reduction in feed consumption of chicks with single heavy infections, followed by an increase in excess of the uninfected controls by 9 days after infection with E. acervulina, an observation repeated in the present study (Fig. 2). Williams (1996) also observed a marked reduction in feed intake of birds infected with E. acervulina during the first week after infection, but did not continue his observations for any longer. 4.3. Virulence of different strains of E. acervulina The experiments described herein were designed to investigate the inherent virulence (see Section 4.1) of a drug-resistant (B) and a drug-sensitive (O) strain of E. acervulina administered to chicks at the same dose. It cannot be decided whether any difference in virulence between these two strains is biologically significant unless the variability recorded among either drug-sensitive or drug-resistant strains in previous studies is taken into account. First the profile of the daily oocyst excretion after infection with each strain was examined (Fig. 1). The double peaks of oocyst production observed for both strains were in each case separated by 1 day as
predicted by Warren and Ball (1967), but the first peak of strain O was on day 5 and that of strain B was on day 6. This difference is probably not important, since Warren and Ball (1967) found that the first peak of oocyst production might occur either on day 5 or on day 6 with the same strain. Long (1967, 1968), Hein (1968) and Joyner (1969) confirmed the occurrence of a double peak of oocyst production by E. acervulina and the variability in its timing. The occurrence of a double peak does not seem to be influenced by the numbers of oocysts administered, since Warren and Ball (1967) observed it following a non-crowded oocyst dose, whilst in the present study and in the others just cited, crowded doses were mainly used (but not always). The difference in timing of peaks during oocyst production in the present study cannot therefore be interpreted as being related to drug-resistance. Second, the link between the magnitudes of oocyst inoculum and excretion of the resulting oocyst progeny was examined. Oocyst production of a given strain of coccidium in repeated tests with the same dose is extremely variable, even when the experimental conditions are identical. Table 6 shows the results of some such tests on drug-resistant and sensitive strains abstracted from the literature. In tests using the same sensitive strain at the same dose, carried out in the same laboratory, the difference between the highest and the lowest RP obtained might be between 1.1 and 10.8 times for E. acervulina; 2.6 times for E. tenella; 1.3 times for E. brunetti; or 1.2 times for E. maxima. Equivalent data for resistant or uncharacterized field strains are 1.1 to 4.8 times for E. acervulina, and 6.3 times for E. tenella. Moreover, there may or may not be an overlap of the range of oocyst productions by drug-sensitive and resistant or uncharacterized field strains of the same species under the same laboratory conditions (Table 6). In short, such comparisons reveal considerable inconsistencies. In the present study, the RP of E. acervulina (B) was 1.4 times that of E. acervulina (O), the difference being not statistically significant. Importantly, that difference also falls within the range of all the possible differences between the mean RPs (1.3–1.6) of the three strains of E. acervulina in the studies of Ball (1966), and between the mean RPs (1.3–2.4) of the three investigated by Norton and Joyner (1980). Furthermore, the RPs of laboratory strains of E.
Table 6 Oocyst production dynamics obtained under standard conditions in various laboratories of different strains of four Eimeria species Strain
Drug-resistant?
Oocysts per bird
Mean oocyst production ( 10 6)
No. of tests
Range of oocyst production per bird ( 10 6)
RP mean (range)
Highest RP as multiple of lowest
Reference
E. E. E. E. E. E. E. E. E. E. E. E. E. E.
O H A W W M H&C W M L W W H1 SM1
No No Yes No No ? No No ? ? No No No Yes
50000 50000 50000 500000 80000 80000 1000000 1000 1000 1000 250000 250000 1000 1000
132.6 167.9 105.7 75.6 206.7 317.2 88.2 110.6 143.4 268.1 13.5 5.5 25.4 21.7
11 2 11 2 2 2 2 3 2 2 2 2 19 18
58.6–261.5 146.6–189.2 41.8–182.8 71.5–79.7 187.9–225.5 295.4–338.9 65.0–111.3 20.8–224.3 49.2–237.5 259.9–276.3 11.9–15.1 4.9–6.1 14.8–38.0 6.9–43.2
2652 (1172–5230) 3358 (2932–3784) 2114 (836–3656) 151 (143–159) 2584 (2349–2819) 3965 (3693–4236) 88 (65–111) 110600 (20800–224300) 143400 (49200–237500) 268100 (259900–276300) 54 (48–60) 22 (20–24) 25400 (14800–38000) 21700 (6900–43200)
4.5 1.3 4.4 1.1 1.2 1.1 1.7 10.8 4.8 1.1 1.3 1.2 2.6 6.3
Ball (1966) Ball (1966) Ball (1966) Dunkley (1968) Joyner (1969) Joyner (1969) Jeffers and Challey (1973) Norton and Joyner (1980) Norton and Joyner (1980) Norton and Joyner (1980) Dunkley (1968) Dunkley (1968) Ball (1966) Ball (1966)
acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina brunetti maxima tenella tenella
RP = reproductive potential.
R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
Species
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
acervulina might be higher or lower than those of field strains (Table 6). For instance, the RPs of two field strains were higher than that of a laboratory strain in one study (Norton and Joyner, 1980), but the RP of Ball’s (1966) resistant strain A was lower than those of the sensitive strains H and O. In the present study the RP of the resistant strain B was higher than that of the sensitive strain O, which was common to this study and Ball’s (1966). Since no clear trends occur amongst the previously published data in Table 6, the present difference between the RPs of the B and O strains cannot be considered to be biologically significant. Third, can a link between virulence and fecundity of strains be demonstrated, considering that the RP may vary in birds given the same inoculum? In Ball’s (1966) study, at 1000 oocysts per bird, the sensitive H1 strain of E. tenella was 1.2 times more fecund than the resistant SM1 strain. At 200,000 oocysts per bird, the mortalities recorded were 77 and 60%, respectively, reflecting a difference of about 1.3 times, slightly greater than that for the fecundity. Comparing two laboratory strains of E. tenella at a dose of 625 sporulated oocysts per bird, Joyner and Norton (1969) found oocyst production of the sensitive H strain to be about 6% greater that of the sensitive W strain, but the W strain caused a statistically significantly lower packed erythrocyte volume, which was counter-intuitive. At the higher doses tested, mortality was 70% for the W strain and 83% for the H strain, which precluded the collection of reliable oocyst production data, so the results obtained overall demonstrated no logical relationships. Since no general association between virulence and fecundity of those strains is demonstrable, the positive association between the greater fecundity and virulence of the B strain in the present study cannot be regarded as important. 4.4. Changes in strain characteristics during selection for resistance Very few studies have addressed changes in strain characteristics during experimental selection for drug resistance. Jeffers and Challey (1973) found that, during selection for clopidol- or quinolone-resistance, several strains of E. acervulina apparently became slightly less virulent, whereas, selection for monensin
or nicarbazin-resistance apparently increased virulence. The changes in virulence during selection were extremely variable in magnitude and direction, were measured by an index combining weight gain and diarrhoea, and moreover were not statistically analysed, so they had no predictive value. Furthermore, when comparing the differences in virulence obtained with some of the same strains in a later study (Jeffers, 1978), the numerical variation in the indices revealed a quite different pattern. Chapman (1976) found that the H strain of E. tenella passaged 12 times in the presence of monensin increased in virulence without developing drugresistance. The greater virulence was not due to increased fecundity in the host. In a further study, Chapman (1978) found no significant differences between the fecundities of lines of E. tenella made resistant to decoquinate, clopidol, robenidine or amprolium and a drug-sensitive line. 4.5. Conclusions The variabilities recorded in previous studies with regard to relationships between oocyst inoculum, oocyst production and virulence are so great that the numerically greater RP, lesions and FCRs resulting from infection with the decoquinate-resistant (B) strain, compared with the sensitive (O) strain, of E. acervulina in the present study must be regarded as having no biological significance. Hence, the present results in the context of the scant pre-existing data indicate that no consistent relationship between drugresistance and virulence in coccidia has been proven. Acknowledgements This work was carried out with the financial support of May & Baker Ltd., now known as Merial. I thank Mrs. A.L. Salmon and Mrs. S.M. Panton for technical assistance, and Professor S.J. Ball for reviewing the original typescript.
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23 Ball, S.J., Warren, E.W., 1965. Treatment of experimental avian coccidiosis with a soluble combination of sulphaquinoxaline and diaveridine. Vet. Rec. 77, 1252–1256. Chapman, H.D., 1976. Eimeria tenella in chickens: studies on resistance to the anticoccidial drugs monensin and lasalocid. Vet. Parasitol. 2, 187–196. Chapman, H.D., 1978. Drug resistance in coccidia. In: Long, P.L., Boorman, K.N., Freeman, B.M. (Eds.), Avian Coccidiosis. British Poultry Science Limited, Edinburgh, pp. 387–412. Chapman, H.D., 1982. Field isolate of Eimeria tenella resistant to arprinocid. Vet. Rec. 110, 540–541. Dickinson, E.M., 1941. The effects of variable dosages of sporulated Eimeria acervulina oocysts on chickens. Poult. Sci. 20, 413–424. Dunkley, M.J.W., 1968. Laboratory trials with buquinolate—a new broad-spectrum coccidiostat for poultry. Vet. Rec. 83, 30–34. Gardiner, J.L., 1955. The severity of cecal coccidiosis infection in chickens as related to the age of the host and the number of oocysts ingested. Poult. Sci. 34, 415–420. Hein, H., 1968. The pathogenic effects of Eimeria acervulina in young chicks. Exp. Parasitol. 22, 1–11. Hodgson, J.N., 1968. A new anti-coccidial drug (M&B 15,497): activity studies against laboratory and field strains of Eimeria. Brit. Vet. J. 124, 209–218. Hodgson, J.N., 1970. Coccidiosis: oocyst counting technique for coccidiostat evaluation. Exp. Parasitol. 28, 99–102. Jeffers, T.K., 1978. Genetics of coccidia and the host response. In: Long, P.L., Boorman, K.N., Freeman, B.M. (Eds.), Avian Coccidiosis. British Poultry Science Limited, Edinburgh, pp. 51–125. Jeffers, T.K., Challey, J.R., 1973. Collateral sensitivity to 4-hydroxyquinolines in Eimeria acervulina strains resistant to meticlorpindol. J. Parasitol. 59, 624–630. Joyner, L.P., 1969. Immunological variation between two strains of Eimeria acervulina. Parasitology 59, 725–732. Joyner, L.P., Long, P.L., 1974. The specific characters of the Eimeria with special reference to the coccidia of the fowl. Avian Pathol. 3, 145–157. Joyner, L.P., Norton, C.C., 1969. A comparison of two laboratory strains of Eimeria tenella. Parasitology 59, 907–913. Long, P.L., 1967. Studies on Eimeria mivati in chickens and a comparison with Eimeria acervulina. J. Comp. Pathol. Therap. 77, 315–325. Long, P.L., 1968. The pathogenic effects of Eimeria praecox and E. acervulina in the chicken. Parasitology 58, 691–700. Long, P.L., Rowell, J.G., 1958. Counting oocysts of chicken coccidian. Lab. Pract. 7, 515–519. Long, P.L., Joyner, L.P., Millard, B.J., Norton, C.C., 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Latina 6, 201–217.
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McDougald, L.R., 1982. Chemotherapy of coccidiosis. In: Long, P.L. (Ed.), The Biology of the Coccidia. Edward Arnold, London, pp. 373–427. McDougald, L.R., 1990. Control of coccidiosis: chemotherapy. In: Long, P.L. (Ed.), Coccidiosis of Man and Domestic Animals. CRC Press, Boca Raton, pp. 307–320. McManus, E.C., Campbell, W.C., Cuckler, A.C., 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54, 1190–1193. Michael, E., Hodges, R.D., 1971. The pathogenic effects of Eimeria acervulina: a comparison of single and repeated infections. Vet. Rec. 89, 329–333. Millard, B.J., 1970. Observations on a drug resistant strain of Eimeria tenella. Res. Vet. Sci. 11, 394–397. Norton, C.C., Joyner, L.P., 1980. Studies with Eimeria acervulina and E. mivati: pathogenicity and cross-immunity. Parasitology 81, 315–323. Preston-Mafham, R.A., Sykes, A.H., 1970. Changes in body weight and intestinal absorption during infections with Eimeria acervulina in the chicken. Parasitology 61, 417–424. Reid, W.M., Johnson, J., 1970. Pathogenicity of Eimeria acervulina in light and heavy coccidial infections. Avian Dis. 14, 166–171. Reid, W.M., Pitois, M., 1965. The influence of coccidiosis on feed and water intake of chickens. Avian Dis. 9, 343–348. Ryley, J.F., 1980. Drug resistance in coccidian. Adv. Vet. Sci. Comp. Med. 24, 99–120. Warren, E.W., Ball, S.J., 1967. Schizogonous stages of Eimeria acervulina Tyzzer, 1929. Nature (London) 214, 929–930. Williams, R.B., 1969. The persistence of drug resistance in strains of Eimeria species in broiler chickens following a change of coccidiostat. Res. Vet. Sci. 10, 490–492. Williams, R.B., 1973. Effects of different infection rates on the oocyst production of Eimeria acervulina or Eimeria tenella in the chicken. Parasitology 67, 279–288. Williams, R.B., 1996. The ratio of the water and food consumption of chickens and its significance in the chemotherapy of coccidiosis. Vet. Res. Commun. 20, 437–447. Williams, R.B., 1998. Analysis of the phenotypes in field populations of Eimeria acervulina expressing dual drug-resistance to decoquinate and clopidol. Avian Pathol. 27, 67–73. Williams, R.B., 2001. Quantification of the crowding effect during infections with the seven Eimeria species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks. Int. J. Parasitol. 31, 1056–1069. Williams, R.B., 2006. Tracing the emergence of drug-resistance in coccidia (Eimeria spp.) of commercial broiler flocks medicated with decoquinate for the first time in the United Kingdom. Vet. Parasitol. 135, 1–14.
Relative virulences of a drug-resistant and a drug-sensitive strain of Eimeria acervulina, a coccidium of chickens R.B. Williams Veterinary Research Division, May & Baker Ltd., Ongar, Essex, UK Received 20 August 2005; received in revised form 3 November 2005; accepted 3 November 2005
Abstract The virulence of a field strain of the chicken coccidian parasite Eimeria acervulina (Boreham I), dually resistant to the chemically unrelated anticoccidial agents decoquinate and clopidol, was compared with that of a drug-sensitive laboratory strain (Ongar) of the same species. Following a single heavy infection (prevented from recycling), both strains exhibited pathogenic effects typical of their species, viz., pathognomonic lesions, adverse effects on body weight gain and feed conversion ratio (FCR), but no mortality. One week after infection, chicks infected with either strain had a statistically significantly worse weight gain than the uninfected control; the Boreham I strain produced more oocysts, and caused slightly more severe duodenal lesions and poorer FCRs than the Ongar strain (all those effects being non-significant). After 3 weeks, there were no significant differences between any cumulative effects of either strain, nor any differences from the uninfected control. However, from 2 to 3 weeks after infection, chicks infected with either strain had a greater feed consumption and growth rate than uninfected chicks. When chicks reared on solid floors were given lighter infections of either strain, which were allowed subsequently to recycle naturally, there were no consistent reductions in weight gains, but feed consumption was higher than that of uninfected chicks. Whatever, the mode of infection, there were no significant differences between the weights of infected and uninfected chicks after 3 weeks, but the FCR of infected chicks was usually poorer than that of uninfected chicks. The difference between the virulences of the Boreham I and Ongar strains was not greater than that between various drug-resistant strains or between various sensitive strains of several Eimeria species recorded in the literature. It is therefore concluded that there was no difference between the virulences of the two strains of E. acervulina that could be attributed to the drug-resistance of one of them. # 2005 Elsevier B.V. All rights reserved. Keywords: Chickens; Coccidiosis; Drug-resistance; Eimeria acervulina; Pathogenicity; Virulence
1. Introduction Despite drug-resistance in poultry coccidia being so well documented, it is still virtually impossible to predict what, in general, is its effect on commercial E-mail address: [email protected].
flocks. Most statements regarding the development of resistance to commercially available drugs in the field are general, anecdotal and second-hand, and are not based on experimental studies (e.g., McManus et al., 1968; Ryley, 1980; McDougald, 1982, 1990). The few records of particular resistance-related coccidiosis outbreaks in the field (e.g., Millard,
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.11.004
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
1970; Chapman, 1982) include no detail that might help to elucidate the relevant circumstances. However, a unique study on the emergence of resistance of coccidia to decoquinate in some of the first broiler flocks that received the drug provided detailed information on coccidial population dynamics, drug-sensitivity testing of isolates of several Eimeria species, and the commercial performances of the affected broiler flocks (Williams, 2006). Although decoquinate-resistance of E. acervulina, E. brunetti, E. maxima and E. tenella developed during rearing of the first flocks to be fed the drug on some UK farms, no adverse effect on their performances was apparent (Williams, 2006). Several factors probably contributed to this unexpected outcome, including initially low levels of exposure to the more pathogenic drug-resistant species when each flock was placed on clean litter, and the stimulation of host immunity by continuous exposure to parasites that survived drug action. A further possibility is that the coccidia that had developed drug-resistance might have become concomitantly less virulent. Because very little has been published on any possible relationship between drugresistance and virulence of coccidia, that hypothesis has now been tested. The virulence of a strain of decoquinate-resistant E. acervulina isolated from one of the flocks monitored by Williams (2006) has been compared with that of a drug-sensitive laboratory strain. The results are assessed in the context of the sparse relevant literature.
2. Materials and methods 2.1. General This work was carried out in the same laboratory, using the same methods and facilities as those documented by Williams (1973). One-day-old male hybrid chicks (Rhode Island Red males Plymouth Rock females) were maintained free from coccidial infection until used at 1-week-old. During experiments the birds were reared in groups of five, in wire mesh-floored or solid-floored cages under continuous lighting in heated rooms, and allowed free access to water and a diet as described by Ball and Warren (1965). Water and feed contained no medication.
The Boreham I strain (B) of E. acervulina was isolated in this laboratory in May 1968 by the method of Williams (1969) from a commercial broiler house during the study reported by Williams (2006). It expressed dual resistance to decoquinate (‘Deccox’ – May & Baker Ltd.) and clopidol (‘Coyden 25’ – Dow Chemical Co.) (Williams, 1998). The laboratory strain of E. acervulina used as a control was the Ongar strain (O) described by Ball (1966), which was isolated in this laboratory in February 1958, well before decoquinate and clopidol became commercially available. It was sensitive to a wide range of contemporarily used anticoccidial drugs (Ball, 1966), including decoquinate (Hodgson, 1968). Infections were administered to chicks as aqueous suspensions of sporulated oocysts, by inoculation directly into the crop with a glass pipette. The oocysts of both strains tested were of the same age, and had been harvested from donor birds ca. 3 weeks before use. Oocysts produced in the faeces of chicks were counted by a modification of the method of Long and Rowell (1958), described by Williams (1973). 2.2. Experimental designs 2.2.1. Single infections with E. acervulina Thirty individually identified chicks per treatment were accommodated in six wire mesh-floored cages. Chicks in treatment 1 were uninfected; chicks in treatment 2 were each infected with 500,000 sporulated oocysts of E. acervulina (B); and chicks in treatment 3 were similarly infected with E. acervulina (O). Such a heavy infection is a ‘‘crowded dose’’ as defined by Williams (2001), and was necessary to produce a significant reduction of chick body weight gain. Twenty individually identified chicks in each of the three treatments were weighed singly 1 day before infection and thereafter weekly for 3 weeks. The remaining 10 chicks per treatment were killed on the fifth (six birds) and the sixth day (four birds) after infection, and the pathognomonic lesions in the duodenum and jejunum were scored as , +, ++ or +++ (negative, slight, moderate or severe). Faeces for oocyst counts were collected daily from under each of the four cages of chicks maintained for weighing, from the third day after infection until no oocysts were detected for two consecutive days. The oocysts produced by the birds in each cage were totalled,
R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
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from which the mean oocyst production per bird was calculated for each strain. The reproductive potential (RP) of each strain was also calculated (the number of oocysts produced by each bird per oocyst administered). Daily feed consumption by the chicks being weighed was measured throughout on a cage basis (four per treatment), and cumulative feed conversion ratios (FCRs) were calculated at the end of each week. FCR = cumulative total of feed eaten (kg)/cumulative total weight gain (kg) of birds in a cage. 2.2.2. Recycling infections with E. acervulina The object of this design was to assess the effect of allowing experimentally infected chicks to reinfect themselves repeatedly by facilitating access to their own faeces, as occurs naturally in the field. A sheet of corrugated cardboard completely covered the wire mesh floor of each cage, so that autoreinfection of infected chicks might occur. Ten individually identified chicks per treatment (in two cages) were used. Chicks in treatment 1 were uninfected; chicks in treatment 2 were each infected with 5000 sporulated oocysts of E. acervulina (B); and chicks in treatment 3 were similarly infected with E. acervulina (O), the doses being intended to mimic a moderate field challenge. Five fresh individual faeces per cage were collected and examined using the method of Hodgson (1970) after 1, 2 and 3 weeks to confirm that recycling had occurred in the infected chicks and that uninfected chicks remained free from coccidia. All chicks were individually weighed 1 day before infection and thereafter weekly for 3 weeks. Daily feed consumption was measured throughout on a cage basis (two per treatment), and cumulative FCRs were calculated for each cage at the end of each week. 2.3. Statistical analyses Methods and results of statistical analyses are given with the experimental results where appropriate.
Fig. 1. The mean daily oocyst production of the Boreham I and Ongar strains of E. acervulina in unmedicated chicks inoculated with 500,000 sporulated oocysts each.
period comprised days 4–13 after infection. The mean total oocyst production per bird for strain B was 116 million 9.8 million S.E. (RP = 232), and that for strain O was 83.5 million 8.5 million S.E. (RP = 167). The difference was not statistically significant by a t-test (two-tailed, P > 0.05). The uninfected controls excreted no oocysts. The mean weight gains of infected and uninfected chicks at weekly intervals during 3 weeks are shown in Table 1. Both strains caused a statistically significant weight gain depression during the first week (twotailed t-test, P < 0.05), but they were not significantly different from each other. From the end of the first week to 3 weeks, there were no significant differences between the weight gains of birds in the infected treatments or between birds in each of them and the uninfected treatment. No birds died. Fig. 2 shows the mean amount of feed eaten per infected chick, expressed relatively to that eaten by uninfected chicks during 3 weeks. The feed intake of infected chicks was reduced until the ninth day after infection but it subsequently exceeded that of Table 1 Weight gains (g S.E.) of chicks in wire mesh floored cages after a single infection of 500,000 sporulated oocysts each of E. acervulina
3. Results
Strain
After 1 week
After 2 weeks
After 3 weeks
3.1. Single infections with E. acervulina
Uninfected Boreham I Ongar
59.1 1.97 50.9 3.56a 50.1 3.56a
145.6 4.82 127.9 7.62 131.3 8.16
230.0 8.05 229.8 10.05 238.9 12.06
Fig. 1 shows the mean daily oocyst production per chick of the two strains of E. acervulina. The patent
a Each statistically significantly less than the controls (P < 0.05), but not significantly different from each other.
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23 Table 4 Weight gains (g S.E.) of chicks with a recycling infection of 5000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
73.1 2.97 75.7 2.42 71.1 2.59
173.0 6.55a 167.7 5.53 152.0 5.71a
278.5 10.96 278.8 8.98 276.5 9.50
a Just statistically significantly different from each other (P = 0.05).
between FCRs of infected and uninfected chicks were much smaller and the FCR of birds infected with strain O was better (lower) than that of uninfected controls. The magnitudes of differences between strain B and strain O after 1, 2 and 3 weeks were somewhat inconsistent. The observed differences were not amenable to valid statistical analysis because of the wide variation within the small numbers of replicate cages. Pathognomonic duodenal lesions, comprising white plaques, occurred in chicks killed 5 or 6 days after infection (Table 3). Strain B caused more severe lesions than strain O, but the difference (after the necessary pooling of cells in the contingency table to construct a 2 2 table) was not statistically significant (Fisher’s Exact test, P > 0.05).
Fig. 2. The mean feed consumption of chicks (g per day) infected with 500,000 sporulated oocysts each of the Boreham I or Ongar strain of E. acervulina, shown relatively to that of uninfected chicks.
Table 2 FCRs of chicks in wire mesh floored cages after a single infection of 500,000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
1.95 2.11 2.18
2.16 2.38 2.36
2.65 2.74 2.59
3.2. Recycling infections with E. acervulina Monitoring of fresh individual droppings for oocysts confirmed that the chicks infected with either strain continued to recycle infections throughout the experiment, and that the control chicks remained uninfected. Table 4 shows the mean weight gains of infected and uninfected chicks at weekly intervals during 3 weeks. Apart from the result with strain O after 2 weeks, which was only just statistically significant, there were no other differences between the weight gains of any of the treatments. No birds died.
uninfected chicks. Before the ninth day, chicks infected with strain O ate more than those infected with strain B. After the eighth day, the reverse was true. The last day of oocyst production was day 13. The mean FCRs of chicks at weekly intervals during 3 weeks are shown in Table 2. Chicks infected with either strain of E. acervulina had markedly poorer (higher) FCRs at 1 and 2 weeks after infection than uninfected chicks. After 3 weeks, differences
Table 3 Numbers of chicks with coccidial lesions of different severities, 5 or 6 days after a single infection of 500,000 sporulated oocysts each of E. acervulina Strain
Uninfected Boreham I Ongar
Severity of lesions on day 5 after infection
6 1 4
Severity of lesions on day 6 after infection
+
++
+++
0 2 2
0 1 0
0 2 0
4 2 1
+
++
+++
0 1 3
0 1 0
0 0 0
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4. Discussion 4.1. Virulence of coccidia
Fig. 3. The mean feed consumption of chicks (g per day) infected with 5000 sporulated oocysts each of the Boreham I or Ongar strain of E. acervulina, subsequently allowed to recycle, shown relatively to that of uninfected chicks.
Overall, infected chicks ate more than uninfected chicks (Fig. 3). However, there was an inexplicably reduced feed consumption relatively to uninfected controls during days 14–16 by birds infected with strain O. Nevertheless, chicks infected with strain B consistently ate more than those infected with strain O. Table 5 shows the mean FCRs at weekly intervals during 3 weeks. As expected from Fig. 3, infected chicks had poorer FCRs than uninfected chicks. As in the previous experiment, after 3 weeks the FCR of chicks infected with strain B was poorer than that of chicks infected with strain O. Differences between treatments were somewhat inconsistent during each time interval and were not amenable to valid statistical analysis because of the wide variation within the small numbers of replicate cages.
Table 5 FCRs of chicks in solid-floored cages, with a recycling infection of 5000 sporulated oocysts each of E. acervulina Strain
After 1 week
After 2 weeks
After 3 weeks
Uninfected Boreham I Ongar
2.10 2.24 2.17
2.59 3.13 3.24
2.93 3.25 3.09
To establish whether expression of drug-resistance might be linked with the capacities of the strains of E. acervulina studied to cause disease, it is first necessary to clarify some terminology. The observed effects of coccidia on chicks may be dealt with under two headings, viz., pathogenesis (the characteristic capacity of an organism to cause disease, and its course of development), and virulence (the disease-producing power, or degree of pathogenicity of an infection). Hence, in the present context, pathogenicity may be regarded as a qualitative property of a species, and virulence as an intraspecific quantitative effect. Whilst the pathogenic effects of coccidia are fairly well understood, the basis of virulence is not so easily established. The virulence of any particular strain of an Eimeria species might be considered to comprise two broad elements, one that may be manipulated and one that is inherent. The difficulty lies in separating the two. It is well known that administering increasingly higher numbers of sporulated oocysts to birds leads to greater virulence (Dickinson, 1941; Gardiner, 1955). The relationship between numbers of oocysts administered and the resulting progeny of oocysts excreted is also well established, and involves a crowding effect at higher oocyst doses (Williams, 1973, 2001). One might, therefore, intuitively expect that a defined oocyst inoculum, the resulting oocyst production and the severity of the pathogenic effects would be interlinked in some predictable way. However, this is not necessarily the case, because of differences between the inherent virulences of coccidial strains. The results of the present study must be considered in that context. 4.2. Pathogenesis of E. acervulina The pathogenic effects of the Boreham I and Ongar strains conformed in general to those usually recognised for E. acervulina (e.g., Joyner and Long, 1974; Long et al., 1976). Thus, there was no mortality, but pathognomonic lesions comprising white plaques were evident in the duodenum and jejunum, with adverse effects on bird weight gain and FCR. Weight gain reduction occurred during the week following the single heavy infection, after which there
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R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
was a compensatory increase in growth rate. This increase was so marked that 3 weeks after infection, infected chicks were as heavy as or heavier than the uninfected controls (Table 1). Such a phenomenon in chicks receiving single infections was not observed by previous workers who terminated their experiments after only 2 weeks (Hein, 1968; Preston-Mafham and Sykes, 1970). Neither did Reid and Johnson (1970) and Michael and Hodges (1971) record complete compensation for weight gain reductions by 4 weeks in chicks infected once. In the case of recycling light infections in the present study, there were no consistent adverse effects on bird weight gain up to 3 weeks after the initial infection (Table 4). Although infected chicks were here able to compensate for weight gain reduction due to heavy E. acervulina infections or to maintain a normal growth rate in the presence of a light recycling infection, they ate more feed during recovery from the single heavy infection (Fig. 2) and during the whole course of the recycling light infection (Fig. 3) than uninfected chicks. Michael and Hodges (1971) also found that chicks recovering from a heavy infection had an increased feed consumption. Reid and Pitois (1965) demonstrated a reduction in feed consumption of chicks with single heavy infections, followed by an increase in excess of the uninfected controls by 9 days after infection with E. acervulina, an observation repeated in the present study (Fig. 2). Williams (1996) also observed a marked reduction in feed intake of birds infected with E. acervulina during the first week after infection, but did not continue his observations for any longer. 4.3. Virulence of different strains of E. acervulina The experiments described herein were designed to investigate the inherent virulence (see Section 4.1) of a drug-resistant (B) and a drug-sensitive (O) strain of E. acervulina administered to chicks at the same dose. It cannot be decided whether any difference in virulence between these two strains is biologically significant unless the variability recorded among either drug-sensitive or drug-resistant strains in previous studies is taken into account. First the profile of the daily oocyst excretion after infection with each strain was examined (Fig. 1). The double peaks of oocyst production observed for both strains were in each case separated by 1 day as
predicted by Warren and Ball (1967), but the first peak of strain O was on day 5 and that of strain B was on day 6. This difference is probably not important, since Warren and Ball (1967) found that the first peak of oocyst production might occur either on day 5 or on day 6 with the same strain. Long (1967, 1968), Hein (1968) and Joyner (1969) confirmed the occurrence of a double peak of oocyst production by E. acervulina and the variability in its timing. The occurrence of a double peak does not seem to be influenced by the numbers of oocysts administered, since Warren and Ball (1967) observed it following a non-crowded oocyst dose, whilst in the present study and in the others just cited, crowded doses were mainly used (but not always). The difference in timing of peaks during oocyst production in the present study cannot therefore be interpreted as being related to drug-resistance. Second, the link between the magnitudes of oocyst inoculum and excretion of the resulting oocyst progeny was examined. Oocyst production of a given strain of coccidium in repeated tests with the same dose is extremely variable, even when the experimental conditions are identical. Table 6 shows the results of some such tests on drug-resistant and sensitive strains abstracted from the literature. In tests using the same sensitive strain at the same dose, carried out in the same laboratory, the difference between the highest and the lowest RP obtained might be between 1.1 and 10.8 times for E. acervulina; 2.6 times for E. tenella; 1.3 times for E. brunetti; or 1.2 times for E. maxima. Equivalent data for resistant or uncharacterized field strains are 1.1 to 4.8 times for E. acervulina, and 6.3 times for E. tenella. Moreover, there may or may not be an overlap of the range of oocyst productions by drug-sensitive and resistant or uncharacterized field strains of the same species under the same laboratory conditions (Table 6). In short, such comparisons reveal considerable inconsistencies. In the present study, the RP of E. acervulina (B) was 1.4 times that of E. acervulina (O), the difference being not statistically significant. Importantly, that difference also falls within the range of all the possible differences between the mean RPs (1.3–1.6) of the three strains of E. acervulina in the studies of Ball (1966), and between the mean RPs (1.3–2.4) of the three investigated by Norton and Joyner (1980). Furthermore, the RPs of laboratory strains of E.
Table 6 Oocyst production dynamics obtained under standard conditions in various laboratories of different strains of four Eimeria species Strain
Drug-resistant?
Oocysts per bird
Mean oocyst production ( 10 6)
No. of tests
Range of oocyst production per bird ( 10 6)
RP mean (range)
Highest RP as multiple of lowest
Reference
E. E. E. E. E. E. E. E. E. E. E. E. E. E.
O H A W W M H&C W M L W W H1 SM1
No No Yes No No ? No No ? ? No No No Yes
50000 50000 50000 500000 80000 80000 1000000 1000 1000 1000 250000 250000 1000 1000
132.6 167.9 105.7 75.6 206.7 317.2 88.2 110.6 143.4 268.1 13.5 5.5 25.4 21.7
11 2 11 2 2 2 2 3 2 2 2 2 19 18
58.6–261.5 146.6–189.2 41.8–182.8 71.5–79.7 187.9–225.5 295.4–338.9 65.0–111.3 20.8–224.3 49.2–237.5 259.9–276.3 11.9–15.1 4.9–6.1 14.8–38.0 6.9–43.2
2652 (1172–5230) 3358 (2932–3784) 2114 (836–3656) 151 (143–159) 2584 (2349–2819) 3965 (3693–4236) 88 (65–111) 110600 (20800–224300) 143400 (49200–237500) 268100 (259900–276300) 54 (48–60) 22 (20–24) 25400 (14800–38000) 21700 (6900–43200)
4.5 1.3 4.4 1.1 1.2 1.1 1.7 10.8 4.8 1.1 1.3 1.2 2.6 6.3
Ball (1966) Ball (1966) Ball (1966) Dunkley (1968) Joyner (1969) Joyner (1969) Jeffers and Challey (1973) Norton and Joyner (1980) Norton and Joyner (1980) Norton and Joyner (1980) Dunkley (1968) Dunkley (1968) Ball (1966) Ball (1966)
acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina acervulina brunetti maxima tenella tenella
RP = reproductive potential.
R.B. Williams / Veterinary Parasitology 135 (2006) 15–23
Species
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acervulina might be higher or lower than those of field strains (Table 6). For instance, the RPs of two field strains were higher than that of a laboratory strain in one study (Norton and Joyner, 1980), but the RP of Ball’s (1966) resistant strain A was lower than those of the sensitive strains H and O. In the present study the RP of the resistant strain B was higher than that of the sensitive strain O, which was common to this study and Ball’s (1966). Since no clear trends occur amongst the previously published data in Table 6, the present difference between the RPs of the B and O strains cannot be considered to be biologically significant. Third, can a link between virulence and fecundity of strains be demonstrated, considering that the RP may vary in birds given the same inoculum? In Ball’s (1966) study, at 1000 oocysts per bird, the sensitive H1 strain of E. tenella was 1.2 times more fecund than the resistant SM1 strain. At 200,000 oocysts per bird, the mortalities recorded were 77 and 60%, respectively, reflecting a difference of about 1.3 times, slightly greater than that for the fecundity. Comparing two laboratory strains of E. tenella at a dose of 625 sporulated oocysts per bird, Joyner and Norton (1969) found oocyst production of the sensitive H strain to be about 6% greater that of the sensitive W strain, but the W strain caused a statistically significantly lower packed erythrocyte volume, which was counter-intuitive. At the higher doses tested, mortality was 70% for the W strain and 83% for the H strain, which precluded the collection of reliable oocyst production data, so the results obtained overall demonstrated no logical relationships. Since no general association between virulence and fecundity of those strains is demonstrable, the positive association between the greater fecundity and virulence of the B strain in the present study cannot be regarded as important. 4.4. Changes in strain characteristics during selection for resistance Very few studies have addressed changes in strain characteristics during experimental selection for drug resistance. Jeffers and Challey (1973) found that, during selection for clopidol- or quinolone-resistance, several strains of E. acervulina apparently became slightly less virulent, whereas, selection for monensin
or nicarbazin-resistance apparently increased virulence. The changes in virulence during selection were extremely variable in magnitude and direction, were measured by an index combining weight gain and diarrhoea, and moreover were not statistically analysed, so they had no predictive value. Furthermore, when comparing the differences in virulence obtained with some of the same strains in a later study (Jeffers, 1978), the numerical variation in the indices revealed a quite different pattern. Chapman (1976) found that the H strain of E. tenella passaged 12 times in the presence of monensin increased in virulence without developing drugresistance. The greater virulence was not due to increased fecundity in the host. In a further study, Chapman (1978) found no significant differences between the fecundities of lines of E. tenella made resistant to decoquinate, clopidol, robenidine or amprolium and a drug-sensitive line. 4.5. Conclusions The variabilities recorded in previous studies with regard to relationships between oocyst inoculum, oocyst production and virulence are so great that the numerically greater RP, lesions and FCRs resulting from infection with the decoquinate-resistant (B) strain, compared with the sensitive (O) strain, of E. acervulina in the present study must be regarded as having no biological significance. Hence, the present results in the context of the scant pre-existing data indicate that no consistent relationship between drugresistance and virulence in coccidia has been proven. Acknowledgements This work was carried out with the financial support of May & Baker Ltd., now known as Merial. I thank Mrs. A.L. Salmon and Mrs. S.M. Panton for technical assistance, and Professor S.J. Ball for reviewing the original typescript.
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