Monensin, Eimeria tenella Infection, and Effects on the Bacterial Populations in the Ceca of Gnotobiotic Chickens D. D. DYKSTRA and W. M. REID Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication July 12, 1977) ABSTRACT Bacteria-free chicks in separate plastic film isolators were inoculated orally with single species of bacteria. Within an isolator, half the birds were fed unmedicated feed and half received feed containing 100 ppmmonensin. With Clostridium perfringens as the established species of monoflora, bacterial counts from the duodenum were 10 4 times lower and counts from the ceca were three times lower in monensin-fed birds compared to unmedicated birds. Infection with Eimeria tenella stimulated an eight-fold increase in the numbers of C. perfringens in the ceca of unmedicated birds but no increase in monensin-fed birds. With Bacteroides sp. or Streptococcus faecalis as monoflora, there was no difference in the cecal or duodenal populations between medicated and unmedicated birds uninfected with coccidia. In contrast to C. perfringens, populations of Bacteroides sp. and S. faecalis in the ceca decreased five to 100-fold in both medicated and unmedicated chicks after infection with E. tenella. Duodenal populations of C. perfringens, Bacteroides sp., and S. faecalis were unaffected by the coccidial infection. INTRODUCTION
Monensin, which has become established as one of the most used anticoccidials during the past five years, is known chiefly for its anticoccidial activity. Users in the field have often reported other beneficial properties including control of necrotic enteritis. Benefits similar to those derived from feeding bacitracin and other antibiotics have been claimed from feeding monensin. Although in vivo antibacterial activity has not been experimentally established from feeding monensin (Elanco, 1974), further study of the strict anaerobes seemed warranted. A number of anaerobes isolated from the chicken intestine were established as monoflora in bacteria-free chickens. One of these, an isolate of Clostridium perfringens, was reduced in numbers in the duodenum and in the ceca as a result of monensin treatment. Furthermore, bacterial counts of C. perfringens, which increased by 8-fold in unmedicated birds after infection with Eimeria tenella, were maintained at a constant level if the birds were being fed monensin. Comparisons were made of bacterial populations with other monoisolates including Bacteroides sp. and Streptococcus faecalis. MATERIALS AND METHODS
Chicks. White Leghorn chick-embryos obtained from a local hatchery were hatched inside sterile, plastic film isolators (Bradley et 1978 Poultry Sci 57:398-402
al, 1967; Dykstra and Reid, 1978). Single strains of anaerobic bacteria were introduced into individual bird in different isolators establishing a monoflora in the intestine. Onehalf of the birds in each isolator were fed University of Georgia broiler starter ration and the other half received the same ration with the addition of 100 ppm monensin. Both rations were packaged in 800 g quantities in polyethylene bags (double-bagged) and sterilized via gamma radiation. Bacteriology. One isolate each of C. perfringens and S. faecalis, and two isolates of Bacteroides sp. (#157 and #670) originally derived from chicken ceca were established in chicks as monoflora. These bacteria were cultured and identified according to Holdeman and Moore (1972). Anaerobic counts were made on roll tubes which contained 2.5% agar in brain-heart-infusion supplemented with yeast extract, vitamin K, hemin, and cystine hydrochloride (BHI+). The isolates were grown for 18 to 24 hr in BHI+ and one ml of the culture was inoculated individually into the crop using a syringe and cannula. Coccidia. Oocysts of a strain of E. tenella, originally isolated by Dr. Allen Edgar, were surface sterilized (Doll et al., 1963) and inoculated via syringe and cannula into the crop after the monoisolate had become established in the ceca. Severity of the coccidial infection was measured by lesion scoring (Johnson and Reid, 1970).
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Collection and Dilution of Cecal Samples. When a bird was sacrificed, the abdominal feathers were picked and the abdomen scrubbed with 95% alcohol. The abdomen was opened aseptically, the ceca removed, and an incision made in the cecal tips to permit the contents to flow into a preweighed dilution tube receiving a continuous flow of 02-free C 0 2 . For duodenal counts, the duodenal loop was removed and contents expressed into a preweighed tube. Dilutions were made and transferred to roll tubes which were allowed to solidify while spinning in a horizontal position. Colonies were counted after a minimum of 5 days.. Experiment 1. Twenty-four bacteria-free birds established in two isolators were inoculated with C. perfringens at seven days of age. One week later all birds in one isolator were inoculated with 50,000 oocysts of E. tenella, while birds in the other isolator received no parasites. Anaerobic counts from the ceca were made from two unmedicated and two medicated birds from each isolator on the day of inoculation with E. tenella and again on days 3 and 6 post-infection (PI). Similar counts were made from the duodenal contents of two medicated and two unmedicated birds from each isolator on days 0 and 6 PI. Experiment 2. Thirty-six bacteria-free birds established in two isolators were inoculated with Bacteroides sp. #157 at six days of age. Anaerobic counts from the ceca were made from an unmedicated and medicated bird from each isolator when birds were 11, 13, and 15 days old. Those days correspond to days —5, —3, and —1 PI of birds infected with coccidia. When 16 days old (day 0 PI), the remaining 12 birds in one isolator were infected with 50,000 oocysts of E. tenella, while birds in the other isolator remained unparasitized. On days 3 and 6 PI anaerobic bacterial counts were made from the ceca of three unmedicated and three medicated birds from each isolator. Experiment 3. In one isolator, 16 birds accidentally became contaminated with S. faecalis, while the 16 birds in this second isolator which had remained bacteriafree were inoculated with Bacteroides sp. #670 at seven days of age. When birds with monoflora of S. faecalis were 13 days old and those with monoflora of Bacteroides sp. #670 were 15 days old, all chicks were infected with 25,000 oocysts of E. tenella. Anaerobic counts of duodenal and cecal contents were made from
3 birds of each treatment (6 total per isolator) before infection, 3 birds on day 6 PI, and 2 birds on day 11 PI. RESULTS Experiment 1. The population of C. perfringens was significantly lower in the ceca of monensin-fed birds compared to unmedicated birds (Fig. 1). In the absence of coccidia, the average number of Clostridia per gram of cecal contents in monensin-fed birds was 4.2 X 10 8 compared to 1.1 X 10 9 in unmedicated birds. Infection with E. tenella resulted in an increase in the clostridial population to 7.8 x 10 9 /g in unmedicated birds on day 6 PI. However, the proliferation of C. perfringens was suppressed in chickens fed monensin (5.8 X 10 8 /g) and did not differ significantly from uninfected, medicated birds (3.7 x 10 8 /g). The population of C. perfringens was significantly greater in duodenal contents of unmedicated birds (3.8 X 10 and 4.1 x 10 7 ) compared to monensin-fed birds (2.8 x 10 3 /g and 2.4 X 10 3 /g) on days 0 and 6 PI, respectively (Table 1). In contrast to the cecal population, the duodenal population in unmedicated and medicated birds did not significantly increase on the 6th day after infection with E. tenella. Experiment 2. The population of Bacteroides sp. #157 was similar in unmedicated and monensin-medicated birds and remained greater
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Monensin, which has become established as one of the most used anticoccidials during the past five years, is known chiefly for its anticoccidial activity. Users in the field have often reported other beneficial properties including control of necrotic enteritis. Benefits similar to those derived from feeding bacitracin and other antibiotics have been claimed from feeding monensin. Although in vivo antibacterial activity has not been experimentally established from feeding monensin (Elanco, 1974), further study of the strict anaerobes seemed warranted. A number of anaerobes isolated from the chicken intestine were established as monoflora in bacteria-free chickens. One of these, an isolate of Clostridium perfringens, was reduced in numbers in the duodenum and in the ceca as a result of monensin treatment. Furthermore, bacterial counts of C. perfringens, which increased by 8-fold in unmedicated birds after infection with Eimeria tenella, were maintained at a constant level if the birds were being fed monensin. Comparisons were made of bacterial populations with other monoisolates including Bacteroides sp. and Streptococcus faecalis. MATERIALS AND METHODS
Chicks. White Leghorn chick-embryos obtained from a local hatchery were hatched inside sterile, plastic film isolators (Bradley et 1978 Poultry Sci 57:398-402
al, 1967; Dykstra and Reid, 1978). Single strains of anaerobic bacteria were introduced into individual bird in different isolators establishing a monoflora in the intestine. Onehalf of the birds in each isolator were fed University of Georgia broiler starter ration and the other half received the same ration with the addition of 100 ppm monensin. Both rations were packaged in 800 g quantities in polyethylene bags (double-bagged) and sterilized via gamma radiation. Bacteriology. One isolate each of C. perfringens and S. faecalis, and two isolates of Bacteroides sp. (#157 and #670) originally derived from chicken ceca were established in chicks as monoflora. These bacteria were cultured and identified according to Holdeman and Moore (1972). Anaerobic counts were made on roll tubes which contained 2.5% agar in brain-heart-infusion supplemented with yeast extract, vitamin K, hemin, and cystine hydrochloride (BHI+). The isolates were grown for 18 to 24 hr in BHI+ and one ml of the culture was inoculated individually into the crop using a syringe and cannula. Coccidia. Oocysts of a strain of E. tenella, originally isolated by Dr. Allen Edgar, were surface sterilized (Doll et al., 1963) and inoculated via syringe and cannula into the crop after the monoisolate had become established in the ceca. Severity of the coccidial infection was measured by lesion scoring (Johnson and Reid, 1970).
398
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MONENSIN, E. TENELLA AND ANAEROBES
Collection and Dilution of Cecal Samples. When a bird was sacrificed, the abdominal feathers were picked and the abdomen scrubbed with 95% alcohol. The abdomen was opened aseptically, the ceca removed, and an incision made in the cecal tips to permit the contents to flow into a preweighed dilution tube receiving a continuous flow of 02-free C 0 2 . For duodenal counts, the duodenal loop was removed and contents expressed into a preweighed tube. Dilutions were made and transferred to roll tubes which were allowed to solidify while spinning in a horizontal position. Colonies were counted after a minimum of 5 days.. Experiment 1. Twenty-four bacteria-free birds established in two isolators were inoculated with C. perfringens at seven days of age. One week later all birds in one isolator were inoculated with 50,000 oocysts of E. tenella, while birds in the other isolator received no parasites. Anaerobic counts from the ceca were made from two unmedicated and two medicated birds from each isolator on the day of inoculation with E. tenella and again on days 3 and 6 post-infection (PI). Similar counts were made from the duodenal contents of two medicated and two unmedicated birds from each isolator on days 0 and 6 PI. Experiment 2. Thirty-six bacteria-free birds established in two isolators were inoculated with Bacteroides sp. #157 at six days of age. Anaerobic counts from the ceca were made from an unmedicated and medicated bird from each isolator when birds were 11, 13, and 15 days old. Those days correspond to days —5, —3, and —1 PI of birds infected with coccidia. When 16 days old (day 0 PI), the remaining 12 birds in one isolator were infected with 50,000 oocysts of E. tenella, while birds in the other isolator remained unparasitized. On days 3 and 6 PI anaerobic bacterial counts were made from the ceca of three unmedicated and three medicated birds from each isolator. Experiment 3. In one isolator, 16 birds accidentally became contaminated with S. faecalis, while the 16 birds in this second isolator which had remained bacteriafree were inoculated with Bacteroides sp. #670 at seven days of age. When birds with monoflora of S. faecalis were 13 days old and those with monoflora of Bacteroides sp. #670 were 15 days old, all chicks were infected with 25,000 oocysts of E. tenella. Anaerobic counts of duodenal and cecal contents were made from
3 birds of each treatment (6 total per isolator) before infection, 3 birds on day 6 PI, and 2 birds on day 11 PI. RESULTS Experiment 1. The population of C. perfringens was significantly lower in the ceca of monensin-fed birds compared to unmedicated birds (Fig. 1). In the absence of coccidia, the average number of Clostridia per gram of cecal contents in monensin-fed birds was 4.2 X 10 8 compared to 1.1 X 10 9 in unmedicated birds. Infection with E. tenella resulted in an increase in the clostridial population to 7.8 x 10 9 /g in unmedicated birds on day 6 PI. However, the proliferation of C. perfringens was suppressed in chickens fed monensin (5.8 X 10 8 /g) and did not differ significantly from uninfected, medicated birds (3.7 x 10 8 /g). The population of C. perfringens was significantly greater in duodenal contents of unmedicated birds (3.8 X 10 and 4.1 x 10 7 ) compared to monensin-fed birds (2.8 x 10 3 /g and 2.4 X 10 3 /g) on days 0 and 6 PI, respectively (Table 1). In contrast to the cecal population, the duodenal population in unmedicated and medicated birds did not significantly increase on the 6th day after infection with E. tenella. Experiment 2. The population of Bacteroides sp. #157 was similar in unmedicated and monensin-medicated birds and remained greater
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