Sensitivity of Eimeria Field Isolates in the United States: Responses of Nicarbazin-Containing Anticoccidials

Nicarbazin (NIC) remains one of the most widely recognized anticoccidial products used by the broiler industry worldwide. For over 50 yr, the product has been known for broad-spectrum anticoccidial activity and resilience in a wide variety of commercial conditions. In his detailed review of NIC, Chapman (1994) suggested that of many compounds introduced for the prevention of coccidiosis in broilers, none could claim a practical life of field utilization as prolonged as that of NIC. In the United States and elsewhere, NIC 125 ppm is commonly used in starter feeds; in this role, it is considered a highly effective anticoccidial with a minimal potential for drug resistance (Bafundo and Jeffers, 1990; Chapman, 1994).

Nicarbazin has also been shown to potentiate the activity of the ionophorous anticoccidials (Callender and Jeffers, 1980), and since the late 1980s, a combination of narasin and NIC (NAR + NIC) has been available for the prevention of coccidiosis. Due to its potentiated effects, lower dosages of this product are used, with 60 to 100 ppm approved in many countries. In the United States, 80 ppm is the most commonly used level of this combination. Commercially, NIC and NAR + NIC have been used in the starter segment of anticoccidial programs, and research has shown positive effects with these products when they are used in this manner (Long et al., 1988; Guneratne and Gard, 1991)

During the introduction of NAR + NIC, however, several investigators (Chapman, 1989; Tamas and Wilks, 1989; Tamas et al., 1991) expressed concerns about the development of resistance by coccidia to the components of the product. In particular, Tamas et al. (1991) speculated that the use of this combination would compromise the effectiveness of NIC. Bafundo and Jeffers (1990) illustrated that there was potential for Eimeria acervulina to develop resistance to NIC and to potentiated NIC combinations (e.g., NAR + NIC) but reported that the same trend was unlikely for Eimeria tenella. These authors recommended limiting the use of potentiated NIC combinations such as NAR + NIC to starter feeds to minimize the risks of resistance development.

Since that time, NIC and NAR + NIC have both been widely used in the United States and other broiler-producing countries. In the past 20 yr, few research studies have been directed toward a better understanding of resistance development when NIC or NAR + NIC are used to prevent coccidiosis at a commercial level.

Thus, the purpose of the work presented herein was to assess overall sensitivity of Eimeria spp. in broilers in the United States to NIC and NAR + NIC, to ascertain whether changes in efficacy of these products have occurred, and to confirm or refute these results with those of contemporary floor pen studies carried out with recent field isolates of coccidia.

 

 

Between 2003 and 2006, litter samples were collected from 26 broiler complexes representing a cross section of commercial operations in the United States. The collection of these samples was not influenced by any specific anticoccidial medication that had been used in previous grow-outs. All of the complexes sampled used typical anticoccidial drug programs involving commonly used ionophores (salinomycin, monensin and narasin, etc.) and chemical products, including NIC and NAR + NIC, among others. However, complexes that had recently used coccidiosis vaccines were not sampled. Additionally, efforts were made to identify collection sites as typical farms that were characteristic of the complex in question. Samples were obtained from the brooding end of each house when flocks were between 21 and 28 d of age. Samples were not collected from problem farms, where consistent, chronic cases of coccidiosis or enteric disease had been diagnosed.

Upon receipt in the laboratory, litter samples were mixed with an equal amount of feed and fed to nonmedicated coccidia-free broilers. The oocysts produced by these birds were purified and sporulated according to standard procedures. These organisms were used to inoculate the birds used in the sensitivity studies.

Male Cobb × Cobb broiler chicks were used in the sensitivity evaluations. These birds were reared in a coccidia-free environment to 12 d of age, when they were weighed and assigned to treatment. Initial cage weights were equivalent for all replicates. Cages were blocked by location in the battery, and assignment of treatments to cages was by use of a random numbers table. Each cage contained 8 birds, and each treatment was replicated 3 times within each experiment. Thus, 24 birds were evaluated per treatment per coccidial isolate. Each coccidial isolate was evaluated in the following treatments: 1) nonmedicated, noninfected; 2) nonmedicated, infected; 3) NIC 125 ppm, infected; and 4) NAR + NIC 80 ppm, infected. Dosages for the anticoccidial products were chosen on the basis of the most common usage levels in commercial practice in the United States. Batteries were maintained with even illumination at a stocking density of 0.19 m2 /bird.

On d 14 of each test, chicks designated to be challenged (treatments 2, 3, and 4) were infected orally with 1 mL of a suspension containing 1 × 105 sporulated oocysts. On d 20 (6 d postinfection), the trial was ended, birds were weighed by cage, and total feed consumption by cage was determined. All birds were killed and lesion scored according to the method of Johnson and Reid (1970). Lesion scores were determined for the upper, middle, and cecal regions of the digestive tract, corresponding to lesions of E. acervulina, Eimeria maxima, and E. tenella, respectively. These data were then used to calculate percentage of reduction in lesions produced by NIC and NAR + NIC for each coccidial isolate. Using the guidelines established by McDougald et al. (1986), these percentages were then used to determine whether each field isolate was sensitive (more than 50% reduction in lesion scores), resistant (less than 30% reduction in lesions), or partially resistant (between 30 and 50% reduction in lesions) to NIC and NAR + NIC.

Mean total lesion scores, weight gain, and feed conversions recorded for each field sample were averaged by treatment for all 26 isolates. Following the recommendations of Chapman (1980), body weight was analyzed to determine whether statistical differences among treatments occurred due to coccidial challenge.

To confirm the findings of the sensitivity tests, 2 floor pen studies were carried out. The pens of chickens used in these tests were reared on used litter that contained contemporary isolates of field coccidia. None of these coccidial isolates were taken from the pool of 26 field isolates described above; however, they were typical field isolates common in the southeastern United States at the time each study was conducted.

The first floor pen test (experiment 2) involved a comparison of NIC and NAR + NIC when each anticoccidial was fed to 9 pens of broilers (60 Cobb × Cobb males per pen) in a 14-d starter program. A nonmedicated treatment was included in this test as a control. A randomized block design was employed. On d 15, anticoccidial medication was changed so that both medicated treatments received salinomycin (55 ppm) to d 28. From d 29 to 42, these treatments received salinomycin (44 ppm). The diets used were corn-soybean meal-based rations that met or exceeded NRC standards. Diets were fed as crumbles (starter feed) or as pellets in all remaining feeds. Dosage levels of the products (NIC 125 ppm or NAR + NIC 80 ppm) were chosen on the basis of the most commonly used levels in the United States; no additional antibiotic medication was provided throughout the trial. All feeds were assayed for the appropriate anticoccidial, and each was found to be within specification. At each feed change and at trial termination, birds and feed were weighed so that average weight gain and feed conversion could be calculated. At these same intervals, 4 birds per pen were examined for the presence of coccidial lesions using the Johnson and Reid (1970) methodology.

In the second floor pen test (experiment 3), NIC 125 ppm and NAR + NIC 80 ppm were compared in a starter program that was fed for 18 d. In addition to the SENSITIVITY TO NICARBAZIN-CONTAINING ANTICOCCIDIALS 1761 by guest on July 26, 2016 http://ps.oxfordjournals.org/ Downloaded from anticoccidials, each treatment contained 50 ppm of roxarsone and 55 ppm of bacitracin. The grower feeds for both treatments (d 18 to 31) were supplemented with 110 ppm of monensin, 50 ppm of roxarsone, and 55 ppm of bacitracin. All finisher feeds contained 11 ppm of virginiamycin. Treatments were administered to a total of 12 floor pens containing 30 male and 30 female Cobb × Cobb broilers. Diet composition and form were similar to the previous trial. Performance responses were determined at d 18 and 38; lesion scores were not recorded during this trial. Analyzed concentrations of all drugs were within the permitted analytical variance.

All data were analyzed using the GLM procedure of SAS (SAS Institute, 2004). Differences among treatments in performance and lesion scores in the sensitivity evaluations were determined by Duncan’s new multiple range test. A probability level of P < 0.001 was used to determine significance. Similar procedures were used to separate treatment means for the floor pen data; however, significance was established at P < 0.05.

The individual results of the 26 field samples of coccidia are presented in Table 1. These results clearly illustrate that all field samples contained E. acervulina, whereas E. maxima and E. tenella were evident in only 4 and 7 samples, respectively. Sixteen of the samples contained a single species (E. acervulina), whereas 9 samples contained mixtures of E. acervulina with E. maxima or E. tenella. Only 1 sample contained all 3 species. Table 1 also indicates that the majority of the coccidial isolates examined were sensitive to NIC with only 4 strains (3 E. acervulina and 1 E. maxima) demonstrating resistance to this anticoccidial; 3 additional E. acervulina isolates were shown to display partial resistance to this product. All E. tenella isolates were sensitive to NIC. In contrast to these findings, the majority of the E. acervulina isolates showed that either resistance (14 isolates) or partial resistance (9 isolates) had developed to NAR + NIC. Similar trends were also shown for E. maxima (3 of 4 resistant) and E. tenella (2 resistant and 1 partially resistant).

Table 2 illustrates the mean total lesion scores produced by these coccidial isolates and shows that medicated feeds significantly reduced lesions compared with the nonmedicated treatment. However, birds receiving NIC had fewer coccidial lesions than those fed NAR + NIC (P < 0.001). Performance data indicated that weight gain and feed conversion for the NIC treatment were significantly improved versus the NAR + NIC treatment (P < 0.001). Performance responses for both medicated treatments fell between the positive and the negative control groups.

The coccidial lesion scores determined at d 14 and 28 of the first floor pen test (experiment 2) are presented in Table 3. Analysis of 14-d lesion scores clearly demonstrated that NIC produced significant reduction in coccidial lesions in all 3 segments of the gut and in average lesion scores. These figures were significantly lower than those produced by NAR + NIC and the nonmedicated control treatment (P < 0.05). At this same interval, NAR + NIC did not reduce upper (E. acervulina) or cecal (E. tenella) lesions compared with the nonmedicated control group (P > 0.05). A similar response was observed for average lesion scores. Because salinomycin (55 ppm) was fed to both treatments from d 15 to 28, statistically similar upper and midintestinal lesions were recorded for medicated treatments at d 28. However, cecal lesions scored at this interval were lower for birds that had received NIC (P < 0.05) than the other treatments. Average lesions measured at d 28 illustrate significant differences among the 3 treatments (P < 0.05); birds that had received NIC had the lowest level of coccidial exposure at that time.

The performance results produced in experiment 3 (Table 5) indicate that by d 18, birds fed NIC had gained more weight and converted feed better than those receiving NAR + NIC (P < 0.05). These differences persisted throughout the grower and finisher periods so that final body weights reflected the same statistical differences established in the starter programs. Comparison of performance results of grower-finisher programs demonstrated no statistical differences between treatments (P > 0.05).

 

Since the introduction of NAR + NIC, few studies have addressed the concept of resistance development to this combination by field coccidia. Although concerns over resistance were expressed by several authors in the years surrounding its introduction (Chapman, 1989; Tamas and Wilks, 1989; Bafundo and Jeffers, 1990; Tamas et al., 1991), the brief reports by Mathis (1999) and Naciri et al. (2003) appear to be the only observations on this subject in subsequent years. As a result, the data presented herein provide the first detailed assessment of possible changes in efficacy of NIC-containing drugs since the late 1980s.

Tamas et al. (1991) expressed concerns that the use of ionophore-NIC combinations would compromise the efficacy of NIC, rendering it ineffective despite its longstanding history of efficacious performance. However, the results of our studies indicate clearly that the large majority of coccidial isolates examined from United States broiler farms were sensitive to NIC when the definitions established by McDougald et al. (1986) were applied to these data. Overall, 81% of the Eimeria strains examined were sensitive to NIC. In addition, 77% of the E. acervulina, 75% of the E. maxima, and all E. tenella strains that were examined were sensitive to the drug. Nicarbazin reduced mean total lesion scores by 74% (Table 2), further supporting the fact that the majority of these field isolates were controlled by NIC. In general, these results are consistent with many other field reports (reviewed by Chapman, 1994) indicating a low level of resistance to NIC in field strains of coccidia.

Conversely, sensitivity to NAR + NIC was rare among these same coccidial isolates, with only 22% showing complete sensitivity to the combination. Of concern was the fact that a very high percentage of E. acervulina (88%) and E. maxima (75%) isolates displayed either resistance or partial resistance to NAR + NIC. The responses of E. tenella indicated that 29% of the isolates were resistant. Analysis of mean total lesion scores revealed that the combination lowered lesion scores by about 35%, a level which was significantly poorer than that of NIC. These results are consistent with brief reports on sensitivity of some coccidial isolates to this combination (Chapman, 1989; Mathis, 1999; Naciri et al., 2003).

 

Chapman (1980) indicated that when sensitive isolates are controlled by medication, weight gain responses are expected to be equivalent to the nonmedicated, noninfected treatments. Because neither medicated treatment in our tests produced such results (Table 2), it is reasonable to assume that some of these field isolates had lost some degree of sensitivity to the drugs tested. This observation is consistent with the lesion score data presented in Table 1 and is to be expected for anticoccidials that are commonly used under field conditions. Despite this observation, the performance responses generated in these studies demonstrated that NIC-fed broilers were significantly heavier and converted feed more efficiently than those receiving NAR + NIC. As with the lesion score data presented previously, these responses support the idea that differences in sensitivity to these anticoccidials currently exist.

Watkins (1997) has taken issue with both of the above methods by suggesting that sensitivity studies do not accurately predict anticoccidial performance under field conditions. To illustrate his point, floor pen trials were conducted that apparently showed little correlation to cages studies with the same anticoccidials. Although we are not aware of other published reports that support this position, we recognize that sensitivity tests involve coccidial exposure techniques that are not normally encountered by commercial broilers. Therefore, it is possible that the severity of infection in cage tests could be greater than that in the field setting, and responsiveness of the drugs under investigation could be less than that encountered in the field. This discrepancy could lead to errors in the interpretation of sensitivity data. Despite the difference in the types of trials used and the apparent pitfalls associated with their interpretation, sensitivity testing may provide some indication of changes within a coccidial population before their manifestation as problems in the field. For example, sensitivity tests demonstrated changes in ionophore sensitivity (Chapman, 1979; McDougald, 1981) long before investigators recognized that resistance to these drugs was characterized by an erosion of field activity rather than a complete loss of effectiveness (Jeffers, 1989). In addition, results of these tests have been reasonable indicators of NIC activity over its many years of usage. As viewed by the production or field specialist, these predictive aspects of sensitivity tests frequently outweigh their shortcomings and may provide some indication of when changes to an apparently effective program may be warranted (Ruff et al., 1997). Contrary to the results of Watkins (1997), we observed a strong correlation between the results of our 2 floor pen trials with the results of the sensitivity tests that were carried out, even if the severity of lesion scores and the effects of coccidial infection on performance were not as severe in floor pens as those produced in the cages. These results show that average lesion scores at d 14 and 28 were significantly lower for NIC than NAR + NIC, and at d 14, NAR + NIC failed to reduce upper intestinal (E. acervulina) and cecal (E. tenella) lesions compared with the nonmedicated treatment. Moreover, significant differences in body weight were evident between medicated treatments irrespective of the time period at which growth was measured (Tables 4 and 5); similar responses were recorded in feed conversion at d 14 (Table 4) and throughout experiment 3 (Table 5). In our opinion, the consistency of results generated in these 2 types of trials lends credence to their accuracy. Furthermore, applying widely accepted definitions of sensitivity, resistance, or reduced sensitivity to these lesion scores (McDougald et al., 1986) and performance data (Chapman, 1980) underscores the fact that coccidial sensitivity to NAR + NIC has changed over the last 20 yr.

Support for the above contention is evident from the review of previous cage studies (Callender et al., 1987) demonstrating that 55 single-species field isolates and 32 mixed-species isolates were completely sensitive to the effects of NAR + NIC. In addition, Long et al. (1988) demonstrated that lesion scores for NAR + NIC 80 ppm in floor pens were equal to or lower than those of NIC 125 ppm; performance responses of NAR + NIC 80 ppm in their tests were significantly greater than those of NIC, as were several suboptimal dosages (20 to 60 ppm of total drug concentration). Likewise, Guneratne and Gard (1991) and Watkins and Bafundo (1993) demonstrated body weight responses for the 2 products were similar to those reported by Long et al. (1988). Thus, data contained herein provide a striking contrast to results of studies carried out during the introduction of NAR + NIC.

 

 

Because the majority of the coccidial strains evaluated in this study have retained sensitivity to NIC, it is reasonable to assume that the loss of activity associated with the NAR + NIC combination is associated with a reduction in effectiveness of the ionophore portion of the combination. This observation is consistent with numerous publications illustrating that ionophore anticoccidials have lost, over time, some degree of effectiveness (Jeffers, 1989; Chapman, 2005). This loss of activity has become more pronounced for the commonly used ionophores in recent years (Mathis, 1999; Bafundo and Hopkins, 2000; Naciri et al., 2003). Indeed, field specialists today clearly recognize the changes in ionophore activity brought about by current overusage of these drugs. Although the initial descriptions of potentiated NIC combinations described activity against coccidia that were resistant to ionophores (Callender and Jeffers, 1980), these studies were carried out 30 yr ago with coccidia that had been exposed to ionophores for no more than 10 yr. Because ionophores have been the primary means by which the poultry industry has controlled coccidia for nearly 40 yr, it is reasonable to assume that further changes in the sensitivity of field coccidia to ionophores have occurred over this time, and these changes could account for the differences presented herein.

Augustine et al. (1987) have investigated ionophore resistance at the cellular level and have demonstrated that sporozoites of ionophore-resistant coccidia do not accumulate as much ionophore as do sensitive organisms. Because ionophores are known to increase the energy requirements of sporozoites (Smith et al., 1981), and NIC is suspected to interfere with energy production by coccidial mitochondria (Wang, 1978), the potentiated effect of these compounds together probably involves additional stress on energy production or utilization, or both, by these parasites. If the process of reduced ionophore uptake by coccidia not fully susceptible to ionophores were to occur in the presence of NIC, the potentiated effect of the ionophore-NIC combination could be diminished. Our results suggest that the loss of activity of NAR + NIC is similar in many respects to the erosion of activity typically observed when ionophore-resistant coccidia develop. That is, reduced efficacy and performance are observed in the presence of normally efficacious levels of the drug

From a long-term perspective, it is important to emphasize that few significant new anticoccidial methodologies are expected in the coming years (Mathis, 2005). This fact, coupled with the understanding that commercial ionophore usage has now expanded to include the control of clostridial enteritis, makes it unlikely that a reduction in ionophore resistance by Eimeria spp. will take place. As a result, it is plausible to propose that the long-term efficacy of NAR + NIC will continue to be adversely affected by the use and overuse of narasin and salinomycin, 2 ionophores separated by only 1 methyl group in chemical structure. Because cross resistance to these ionophores has been described (Chapman, 1986, 1989), and Augustine et al. (1987) have demonstrated the mechanism for its occurrence, this factor is likely to influence the efficacy of NAR + NIC in the future. Consideration of these multiple effects should become part of prudent anticoccidial program planning now and in the years ahead.

 

 

Augustine, P. C., C. K. Smith, H. D. Danforth, and M. D. Ruff. 1987. Effect of ionophorous anticoccidials on invasion and development of Eimeria: Comparison of sensitive and resistant isolates and correlation with drug uptake. Poult. Sci. 66:960–965.

Bafundo, K. W., and B. Hopkins. 2000. Laboratory and field observations on the effectiveness of selected ionophores with reference to continuous usage practices. Poult. Sci. 79(Suppl. 1):106. (Abstr.)

Bafundo, K. W., and T. K. Jeffers. 1990. Selection for resistance to monensin, nicarbazin and the monensin plus nicarbazin combination. Poult. Sci. 69:1485–1490.

Callender, M. E., D. J. Donovan, L. V. Tonkinson, and R. F. Shumard. 1987. Dose titration of the anticoccidial combination of narasin and nicarbazin. Poult. Sci. 66(Suppl. 1):74. (Abstr.)

Callender, M. E., and T. K. Jeffers. 1980. Anticoccidial combinations comprising nicarbazin and the polyether antibiotics. US Patent No. 4,218,438. Eli Lilly and Co., assignee.

Chapman, H. D. 1979. Studies on the sensitivity of recent field isolates of Eimeria maxima to monensin. Avian Pathol. 8:181–186.

Chapman, H. D. 1980. Studies on the sensitivity of field isolates of Eimeria maxima to combinations of anticoccidial drugs. Avian Pathol. 9:67–76.

Chapman, H. D. 1986. Isolates of Eimeria tenella: Studies on resistance to ionophorous anticoccidial drugs. Res. Vet. Sci. 41:281–282.

Chapman, H. D. 1989. Field isolates of E. tenella: Sensitivity to diclazuril, maduramicin, narasin, salinomycin and a mixture of nicarbazin/narasin. Pages 323–326 in Coccidia and Intestinal Coccidiomorphs: Proceedings of the Vth International Coccidiosis Conference, Tours, France. Institut National de la Recherche Agronomique, Paris, France.

Chapman, H. D. 1994. A review of the biological activity of the anticoccidial drug nicarbazin and its application for the control of coccidiosis in poultry. Poult. Sci. Rev. 5:231–243.

Chapman, H. D. 2005. Perspectives for the control of coccidiosis in poultry by chemotherapy and vaccination. Pages 99–103 in Proceedings of the IXth International Coccidiosis Conference, Foz de Iguassu, Parana, Brazil. Apinco Foundation of Poultry Science and Technology, Campinas, São Paulo, Brazil. Guneratne, J. R. M., and D. I. Gard. 1991. A comparison of three continuous and four shuttle anticoccidial programs. Poult. Sci. 70:1888–1894.

Jeffers, T. K. 1989. Anticoccidial drug resistance: A review with emphasis on the polyether ionophores. Pages 295–309 in Coccidia and Intestinal Coccidiomorphs: Proceedings of the Vth International Coccidiosis Conference, Tours, France. Institut National de la Recherche Agronomique, Paris, France. Johnson, J., and W. M. Reid. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30–36. Long, P. L., J. Johnson, and M. E. McKenzie. 1988. Anticoccidial activity of combinations of narasin and nicarbazin. Poult. Sci. 67:248–252. Mathis, G. F. 1999. Anticoccidial sensitivity of recent field isolates of chicken coccidia. Poult. Sci. 78(Suppl. 1):116. (Abstr.) Mathis, G. F. 2005. Poultry coccidiosis control: From now to the next coccidiosis conference. Page 3 in Proceedings of the IXth International Coccidiosis Conference, Foz de Iguassu, Parana, Brazil. Apinco Foundation of Poultry Science and Technology, Campinas, São Paulo, Brazil. McDougald, L. R. 1981. Anticoccidial drug resistance in the United States: Polyether, ionophorous drugs. Avian Dis. 25:600–609. McDougald, L. R., L. Fuller, and J. Solis. 1986. Drug sensitivity of 99 isolates of coccidia from broiler farms. Avian Dis. 30:690–694.

Naciri, M., K. DeGussem, G. Fort, N. Bernardet, F. Nerat, and A. M. Chausse. 2003. Utility of anticoccidial sensitivity tests (ASTs) in the prevention of chicken coccidiosis. Br. Poult. Sci. 44:826–827.

Ruff, M. D., H. D. Danforth, and G. C. Wilkins. 1997. Interpreting results of anticoccidial testing. Page 53 in Control of Coccidiosis into the Next Millennium: Proceedings of the VIIth International Coccidiosis Conference. Institute for Animal Health, Compton, Newbury, Berks, UK. SAS Institute. 2004. SAS User’s Guide. SAS Institute Inc., Cary, NC.

Smith, C. K., R. B. Galloway, and S. L. White. 1981. Effect of ionophores on survival, penetration and development of Eimeria tenella sporozoites in vitro. J. Parasitol. 67:511– 516.

Tamas, T., K. D. Schleim, and G. Wilks. 1991. Development of resistance against amprolium, nicarbazin, maduramicin, lasalocid and Maxiban. Poult. Sci. 70(Suppl. 1):119. (Abstr.)

Tamas, T., and G. Wilks. 1989. Development of resistance against narasin and narasin + nicarbazin. Poult. Sci. 68(Suppl. 1):146. (Abstr.)

Wang, C. C. 1978. Biochemical and nutritional aspects of coccidia. Pages 135–184 in Avian Coccidiosis: Proceedings of the 13th Poultry Science Symposium. P. L. Long, K. N. Boorman, and B. M. Freeman, ed. British Poultry Science Ltd., Edinburgh, UK.

Watkins, K. L. 1997. Are sensitivity test results good predictors of anticoccidial field efficacy? in Control of coccidiosis into the next millennium. Page 53 in Proceedings of the VIIth International Coccidiosis Conference. Institute for Animal Health, Compton, Newbury, Berks, UK.

Watkins, K. L., and K. W. Bafundo. 1993. Effect of anticoccidial programs on broiler performance. J. Appl. Poult. Res. 2:55–60.