METHOD OF EVALUATING THE EFFICIENCY OF ANTICOCCIDIAL DRUGS IN FLOORPEN TRIALS WITH MULTIPLE INFEED INFECTION VERSUS SEEDING MODEL P. Yvore, J.P. Raynaud, L. Conan, Muriel Naciri, M. Virat To cite this version: P. Yvore, J.P. Raynaud, L. Conan, Muriel Naciri, M. Virat. METHOD OF EVALUATING THE EFFICIENCY OF ANTICOCCIDIAL DRUGS IN FLOORPEN TRIALS WITH MULTIPLE IN FEED INFECTION VERSUS SEEDING MODEL. Annales de Recherches Vétérinaires, INRA Editions, 1980, 11 (1), pp.99106. <hal00901253> HAL Id: hal00901253 https://hal.archivesouvertes.fr/hal00901253 Submitted on 1 Jan 1980 HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
METHOD OF EVALUATING THE EFFICIENCY OF ANTICOCCIDIAL DRUGS IN FLOORPEN TRIALS WITH MULTIPLE INFEED INFECTION VERSUS «SEEDING» MODEL P. 1YVORE J.P. RAYNAUD 2 L. 3CONAN Muriel NACIRI 1 M. 2VIRAT Il.N.R.A., Laboratoire de Parasito%gie, 37380Nouzilly, France ZAgricultural Research and Deve%pment Station, Pfizer lnternational, 37400 Amboise, France 3/.N.R.A., Station du Magneraud, SaintGeorges du Bois, 17700 Surgères, France Résumé METHODE DE MESURE DE L EFFICACITE D ANTICOCCIDIENS. Les expérimentations au sol (floor pen) permettent l estimation de l efficacité des anticoccidiens. Par la méthode d infestation décrite, c estàdire l ingestion d un aliment contaminé durant cinq jours consécutifs, il est plus facile de contrôler la contamination des animaux que par la méthode des «seeder birds» et l anticoccidien a une double action : il protège les animaux et en même temps diminue la conta un anti mination de l environnement. La présence d un lot expérimental non infecté et traité par coccidien rend possible de juger des performances normales des animaux. En plus des lésions, la coloration plasmatique et les hématocrites sont d excellents critères pour évaluer l incidence du parasitisme. Un plan expérimental est proposé. According to Raines (1978), data from three phases of experiment are necessary to evaluate the efficacy of anticoccidial drugs : battery experiments, floor pen experiments and fiel trials. In battery experiments it is possible to work with a small number of birds and to check their infection very closely. Consequently, a greater number of treatments can be included with different experimental criteria : zootechnical results ; lesion index, symptomatological criteria (blood drops, hematocrits), action on digestive function (serum coloration) and development of parasitic population (oocyst excretion and sporogony). However, while such tests may demonstrate activity, they do not necessarily provide reliable predictions of efficacy in practice. In floor pens, birds undergo a permanent infection depending on the parasitic level of the environment. The effect of anticoccidial drugs is twofold : protection of the animals and diminution of environmental contamination. Cover (1970) considers that floorpen experiments are essential because they simulate practical conditions. These methods have been described by Reid et al. (1969) and by Mitchell and Scoggins (1970). Callender and Shumard (1969), Gard et al. (1969) recommend the use of suspended «seeder birds». More recently, a variation of this method of
reliable the there in infection was suggested (Ruff et al., 1976) ; these authors use either a direct contamination by suspended «seeder birds», or an indirect contamination between experimental pens by worker s shoes. Although the «seeding» method is attractive and gives good results, it has the following drawbacks : data is not available on the litter contamination by the «seeder birds» as the multiplication potential differs according to the parasitic species. Good results are difficult to obtain with strains whose pathogenicity is not well known ; lesion index of Johnson and Reid (1970) is a very important and reliable criterion, but it is sometimes difficult to apply in presence of chronic lesions of the intestinal tract ; is no «non infected control» due to the practical inability to avoid a light accidental contamination by animal handlers and technicians or by the environment on the so called «non infected control animals» ; this method, 15day old animals are introduced into an environment that has been artificially contaminated at a relatively high level whereas under natural conditions this level is lower at the start of breeding. Anticoccidial drugs have a double action : they protect the animals and limit environmental contamination, We have tried to define a methodology that is easy to realize by modifying the method of contamination, by using new experimental criteria and with the presence of a pseudo «non infected control» group. fxperimental Method 1 Experimental design Sexed Hubbard birds are bred during 56 days on a litter of wood shavings in 24 pens in two contiguous rows. The experiments take place in a dark building. During the 1st experiment, artificial light was provided nonstop during the 1st week, and 16 hours per day during the rest of the breeding period ; in the 2nd experiment, artificial light was provided nonstop during the entire breeding period. At the start of the 1st experiment 55 animals were placed in each pen ; on the 10th day this number was reduced to 50. At the end of the breeding period, the density was 6.7 animals per square meter. In the 2nd experiment, the density was increased to 15 square meter by reducing the surface of the pens and by increasing the number of birds per pen. Sexes were segregated. Feed was provided ad libitum. From Day 1 to Day 28 a «starter» feed was given as a mash to facilitate the preparation of the contaminated feed. From Day 29 to Day 56, a «grower» feed was given in pelleted form (Table 11. The birds were slaughtered at 56 days of age.
2 Experimenfal groups Only 3 experimental groups are discussed here, the other concerned the study of the activity of a new anticoccidial drug. The three groups are : T1 : Non artificially infected control, treated with anticoccidial at normal concentration T2 : Artificially infected non treated control T3 : Artificially infected, treated Monensin at 100 ppm Each group comprised 4 pens, 2 of males and 2 of females. The anticoccidials were given continuously in feed including in the infecting feed. «non infected The T1 group is the pseudo control». Infection is only accidental, probably less severe and occurring later than in the other groups. The fact that the strains of coccidia were sensitive to the anticoccidial used to treat this group reduced the consequences of accidental infection. This group makes it possible to have an idea of the production potential of animals under the specific experimental conditions. 3 Method of infection The experimental infection was carried out on 5 consecutive days in the 1st experiment, and during 2 periods of 5 consecutive days in two consecutive weeks in the 2nd experiment. The birds were 13 days of age at first infection. The animals received each days a feed containing oocysts of three species of coccidia, the average dose per animal and per day was 15,000 oocysts of Eimeria tenella, 100,000 of E. acervulina and 20,000 of E. necatrix. The infecting feed was prepared each morning. In the first experiment, drinking water was cut the evening before each infection when artificial light was cut off. In the second experiment, water was maintained. The quantity of infecting feed distributed per animal and per day was 7.5 g, 2.5 g of mash and 5 ml of oocyst suspension. The feed was placed in round plastic dishes 24 cm in diameter. Non infected feed was no longer available to the animals when the infecting feed was given. After 2 h, about 250 ml of water was added to the dishes. Four hours after the start of administration of infecting feed, normal feed and drinking water were again available. 4 Experimental criteria Zootechnical criteria Animals were individually weighed at 1, 10, 28 and 56 days of age. Feed consumption per pen was measured on these same dates. Feed conversion indices obtained are only relative owing to manipulations and modification in numbers of birds. Comparative examination between groups are however possible. Lesion index and biochemical criteria At 20 and at 27 days of age, five animals are
taken from each pen, i.e., 20 animals per treatment. These dates correspond with the 7th and 14 th days after start of infection. For each animal the following parameters were measured : weight, hematocrit, serum coloration and lesion index. To measure hematocrit, blood was taken from the wing vein in a heparinated microtube. This criterion reveals the extent of hemorrages which are essentially caused bye. tenella and partially by E. necatrix. Serum coloration is measured by direct reading on the spectrophotometer at 480 nm with serum from blood taken by intracardiac puncture. Ruff, Reid and Johnson (1974) recommend an extraction by acetone before spectrophotometric reading. This technique is better : only the carotenoids are measured, and some serums which are cloudy are difficult to read. We have retained direct reading because it is simple and speedy to carry out. Discoloration observed in the reading at Day 20 is due in large part to E. acervulina which modifies the intestinal absorption of carotenoids (Yvore and Mainguy, 1972), and in part to E. tenella and E. necatrix which can be responsible at this time for a slight serum discoloration. At Day 28 E. acervulina and E. necatrix are responsible for serum discoloration. The chronic lesions disturb the intestinal absorption. Lesions are scored in accordance with Johnson and Reid (1970) index in regard to three intestinal segments : upper small intestine, middle small intestine and cecum. In our experiments, it was not necessary to examine the lower part of the small intestine because the Eimeria species used do not cause lesions there. Analysis of the results Analysis of variance (Fisher test) and comparison of means (Duncan test) were performed on all criteria except for the lesion index. Seeding experiment In parallel with the infeed multiple infection experiment, an experiment was carried out with direct «seeder» birds. Animals were placed in suspended cages. They are infected and eliminated 10 days later. Five days after their elimination the experimental birds are given access to the contaminated area. At that time they were 15 days of age. The infection was carried out with the same coccidial strains as in the infeed infection experiment. The density at end of the breeding period is 15 per square meter. Results 1 Mortality (coccidiosis) With the infeed infection technique, in
spite of very serious symptoms, no animal died of coccidiosis in the first experiment. In the second experiment, mortality in the infected non treated group was 14.2 %. This was entirely due to the first week of infection. With the «seeder» birds technique, mortality was 29 percent. 2 Control of animal infection In the infeed infection model, the infection was evaluated 7 and 14 days after the start of infection on slaughtered animals. Figures 1 and 2, and table 2 summarise the results obtained. Figure 1 also shows the lesion score index in the «seeding» experiment at Day 23. Seven days after first infection (Day 20), the lesion indices (figure 1) in the T2group (infected non treated) are high. It can be noted that the most severe lesions are due to E. acervulina (upper small intestine) and to E. tenella (cecum). However, lesions due to E. necatrix (middle small intestine) are less extensive. The hematocrit and the serum discoloration are low at a highly significant level (table 2, fig. 21. In the T1group (non infected treated), lesions are practically inexistant and hematocrits are at normal value. In the T3group (treated Monensin 100 ppm), the lesion indexes are low. The hematocrit values are not significantly different from those of the T1group. In the first experiment, a slight serum discoloration can be noted compared to the non infected treated group. A comparison of the lesion indexes at Day 20 and Day 23 for the Monensin treated groups is interesting. These indexes are rather similar in the three experiments. In the two «infeed» experiments, this index is higher than that of the T1group (non infected treated). This shows the importance of having a pseudo non infected control. Fifteen days after first infection (Day 27), the lesions are less severe but they persist especially in the cecum in the infected non treated group. The hematocrit is normal, the hemorrages have disappeared. Some serum discoloration due to chronic intestinal lesions can still be observed. In the other two groups, values for hematocrits and serum discoloration are normal. 3 Zootechnical results Table 3 shows average weight gains and feed conversion data during the period immediately after the infection (D 10 to D 29), and during the whole breeding period (D 1 to D 561. 3. 1. «Seeding experiment» In this experiment the severe infection induced a highly significant diminution of weight gain between Day 10 and Day 28. The weight gain of the infected non treated group is 30 percent lower than that of the treated group. During the period from Day 1 to Day 56 there is no significant difference between the two groups but the feed conversion index shows a highly significant difference ( + 4.4% for the infected non treated group). 3.2. rr lnfeed» contamination model In the first experiment, during the period Day 10 to Day 29, the infection reduced significantly the weight gain by about 29 percent without mortality. In the second experiment, the reduction among surviving animals is about 37 percent. Moreover, in both experiments, the parasitism increased the feed conversion index (P < 0.01). ). The monensin did not completely eliminate the reduction of weight gain in the first experiment. In the second experiment, a slightly lower weight gain than the non infected treated can be observed. In both experiments, the feed conversion data were normal for the monensin treated group. During the total breeding period (Day 1 to Day 56), for the infected non treated group,
the parasitism induced a lower weight gain only in the first experiment, but in both experiments the feed conversion index is higher. The monensin treatment allowed normal weight gains and normal feed conversion indexes in both experiments (no difference with non infected treated group). Discussion 1. Method of infection The infeed infection method makes the infection of the animals more convenient (1 person during 2 hours per day to infect 1,200 birds). It seems preferable to use two small dishes rather than one big one. The animals quickly form two groups, they readily approach infected feed, and they do not stand on the dishes ; this avoids direct contamination of the litter by their feet. This contamination is thus practically entirely indirect through the animals, and the anticoccidials modify this contamination by their action on parasitic multiplication and sometimes on the evolution of oocysts (sporogony). Cutting the drinking water supply a few hours before giving the infecting feed is not necessary to encourage the birds to consume this feed. In addition, it disturbs the chicks to a greater or lesser extent. Individual results show a good homogeneity of infection in the pens. With this method of infection, we control perfectly the number of oocysts administered and the proportions of each species. This makes it possible to work more easily with newly isolated strains whose pathogenicity and rate of multiplication are not perfectly known. We think it useful to infect the animals on several consecutive days with medium doses. A single massive dose does not reproduce normal infection under natural conditions in breeding units. However, one period only of 5 days is sufficient. In the second experiment, the second 5day period did not add anything. At this time the animals have already begun to excrete oocysts. They have already started to infect themselves naturally from the litter. 2. Animal density Animal density seems to be an important factor. In the two experiments carried out with 15 animals per square meter, mortality was higher than in those with a lower density. The importance of this factor was already indicated by Clarke et a/. (1978) who recommend high densities up to 22 bnimals per square meter. It has been noted that under natural breeding conditions, coccidial problems are particularly important in breeding where density is high. Moreover, a density of 15 animals per square meter reproduces better practical field conditions. 3. Interest of a «Non lnfected Treated Control u group t T 1) J The presence of a non infected treated group IT1) seems useful to us. In our experiments, this group made it possible to demonstrate a significant difference in weight gain with the infected monensin treated group (T3) during the days immediately after the infection (Day 10 to Day 29). This difference had disappeared on the day of slaughter (D 56). Without a T1 group, only the anticoccidial action of monensin is demonstrated. When this T1 group is present, we also demonstrate that monensin permits good final performances. 4. Criteria to evaluate parasitism In regard to the experimental criteria, we think that the lesion score is essential on account of its specificity. It makes it possible to evaluate animal infection and the pathogenicity of the coccidial strains. However, its use could be restricted to this evaluation. In this case, lesions would only be scored in the infected non treated group. Moreover, we think that it is sufficient to study the incidence of infection at only one time : 7 days after start of infection. Another evaluation 14 days after the first infection necessitates the slaughter of other animals, modifies the composition of the pens, and does not add any useful information concerning the parasitism and the effect of the anticoccidials. It should also be noted that scoring of lesions at this stage is more difficult and less precise than at the 7th day after start of infection. Data on the hematocrit and the serum co%ration are easier and faster to obtain than the lesion index. There is a close correlation between cecal lesions and hematocrits, and between acute or chronic intestinal lesions and
Like It Contamination The The serum coloration. Evaluation of these criteria is not subjective. So that the experiment plan will remain sufficiently sensitive, it is necessary to slaughter 5 animals per pen (for 4 pens per treatment, 20 animals per treatment). Taking into account the similitude of the results between the classical «seeding» technique and the new «infeed» infection technique, we can conclude that the latter method permits the evaluation of the efficacy of a new anticoccidial. Conclusions In our opinion ; the experiment method described here has the following advantages : the «seeder bird» technique, this is a method for floor pen experiment. Conditions are close to practical breeding conditions. is easy to check infection of the animals (number of oocysts infected and percentage of each species). of the environment is ensured almost exclusively by the experimental animals and influenced by the anticoccidials. non infected treated group can be considered as a real non infected control group. Accidental infection is inevitable but discreet, and the activity on the coccidia of the anticoccidial used is known. experimental criteria suggested (hema
tocrits, and serum coloration) are easy to evaluate, not subjective, and easily subjected to analysis. Nevertheless, we consider that lesion scoring is indispensable. It could be interesting to study litter contamination. Hodgson (1970) suggested methods of sampling, examination and expression of results. It would be interesting to study simultaneously the number of oocysts present and their sporulation percentage. This study could be made from the 5th week of breeding on. Taking into account the means at our disposal, Reid ( 1975, 1978) expresses doubts about the worth of this criterion to evaluate the efficacy of an anticoccidial. Studies would be necessary to improve the methods and to codify them. The main criticism of this new cc infeed» infection method compared to the cc seeder birds» method is that the effect of the infection is evident during a shorter period of time. The animals are ill during 2 weeks only. Floor pen experiments are only the second phase of testing, and they are completed by field trials, phase 3. They are essential to correctly evaluate the efficacy of an anticoccidial on condition that the experimental groups are mastered sufficiently, even if this implies a breakaway from natural conditions of breeding. A possible experiment work is suggested (table 4). Accepted for publication September 26th 1979. Summary The evaluation of the efficacy of anticoccidials requires floor pen experiments. With the method of infection suggested, i.e., the ingestion of an infecting feed during 5 consecutive days, it is easier to check animal contamination than with the «seeder birds» method, and the anticoccidial has a dual action, it protects the animals and at the same time diminishes the environmental contamination. The presence of a non experimentally infected group, treated by an anticoccidial drug makes it possible to judge normal performances of the animals. In addition to lesion scorings, serum coloration and hematocrits are excellent criteria to evaluate the incidence of parasitism. An experimental work plan is suggested. References CALLENDER M.E., SHUMARD R.F., 1969. The use of different infection systems for evaluating the efficacy of an anticoccidial compound in chickens housed in floor pens. Poult. Sci., 48, 19721973. CLARKE M.L., BENTLEY E.J., POLLOCK K.M., 1978. Evaluation of anticoccidial drugs under field conditions. /n Long P.L., Boorman K.N., and Freeman B.M., Avian coccidiosis pp. 347373, British Poultry Science Edinburgh. COVER M.S., 1970. Coccidiosis : introduction to design and execution of floorpen experiments. Expl. Parasit, 28, 63. DANFORTH H.D., RUFF M.D., REID W.M., JOHNSON J., 1977. Anticoccidial activity of salinomycin in floorpen experiments with broilers. Poult. Sci, 56, 933938. GARD D.I., YOUNG D.C., CALLENDER M.E., 1969. Simulating field conditions to evaluate coccidiostat. Poult. Sci., 48, 1811. HODGSON J.N., 1970. Coccidiosis : oocyst counting technique for coccidiostat evaluation. Expl. Parasit., 28, 99102. JOHNSON J., REID W.M., 1970. Anticoccidial drugs : lesion scoring techniques in battery and floorpen experiments with chickens. Expl. Parasit., 28, 3036. MITCHELL G.A., SCOGGINS R.W., 1970. Avian Eimeria technique : suspended seeder cage. Expl. Parasit., 28, 8789. RAINES T.V., 1978. Guidelines for the evaluation of anticoccidial drugs. /n Long P.L., Boorman K.N., Freeman B.M., Avian coccidiosis, pp. 339346, British Poultry Science, Edinburgh. REID W.M., 1975. Relative value of oocyst counts in evaluating anticoccidial activity. Avian Dis., 19, 802811. REID W.M., 1978. Prospects for the control of coccidiosis. /n Long P.L., Boorman K.N., Freeman B.M. Avian Coccidiosis, pp. 501526, British Poultry Science, Edinburgh.
REID W.M., BREWER R.N., JOHNSON J., TAYLOR E.M., HEDGE K.S., KOWALSKI L.M., 1969. Evaluation of techniques used in studies on efficacy of anticoccidial drugs in chickens. Am. J. Vet. Res., 30, 447459. RUFF M.D., REID W.M., JOHNSON J.R., 1974. Lowered blood carotenoid levels in chickens infected with coccidia. Poult. Sci., 53, 18011809. RUFF M.D., REID W.M., RAHN A.P., 1976. Efficacy of different feeding levels of monensin in the control of coccidiosis in broilers. J. Am. Vet. Ass., 37, 963967. YVORE P., MAINGUY P., 1972. Influence de la coccidiose duod6nale sur la teneur en carot6noides du serum chez le poulet. Ann. Rech. V6t., 3, 381387.