Efﬁcacy of In-Feed Tylosin Phosphate for the Treatmentof Necrotic Enteritis in Broiler ChickensJ. Brennan,* G. Moore,†,1S. E. Poe,† A. Zimmermann,† G. Vessie,‡ D. A. Barnum,§ and J. Wilson||*Shur-Gain Agresearch, R.R. 3, Burford, Ontario, Canada, N0E IA0; †Elanco Animal Health, Division of Eli Lilly and Co.,Greenﬁeld, Indiana 46140; ‡Elanco, Division Eli Lilly Canada Inc., Research Park Centre, 150 Research Lane, Suite 120,Guelph, Ontario, Canada N1G 4T2; and Departments of §Pathobiology, and ||Population Medicine,Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1ABSTRACT The efﬁcacy of tylosin phosphate for thetreatment of necrotic enteritis (NE) was investigated in aﬂoor pen study of 2,000 broiler chickens. A model inwhich Clostridium perfringens was administered in thefeed on Days 14 to 16 was used to initiate an outbreakof NE. Treatments, allocated at the pen level in a random-ized complete block design, consisted of ﬁve levels oftylosin phosphate (0, 50, 100, 200, or 300 ppm) adminis-tered in the feed on Days 15 to 22, following the identiﬁ-cation of an outbreak of NE on Day 15. Mortality due toNE was signiﬁcantly reduced (P < 0.05) for medicatedbirds at all dose levels of tylosin phosphate compared to(Key words: tylosin phosphate, necrotic enteritis, broiler chicken, efﬁcacy)2001 Poultry Science 80:1451–1454INTRODUCTIONNecrotic enteritis (NE), caused by the Gram-positivebacillus Clostridium perfringens is an important disease ofbroiler chickens, causing signiﬁcant morbidity and mor-tality worldwide. The disease frequently occurs as out-breaks characterized by depression, anorexia, and suddendeath. Control of this condition is dependent upon a vari-ety of elements including reducing exposure to potentialdietary risk factors, reduction of concurrent enteric infec-tions, particularly coccidiosis, and the use of feed addi-tives with activity against C. perfringens (Ficken andWages, 1997).Although studies have demonstrated the efﬁcacy ofcertain drugs in the prevention of NE (Long and Truscott,1976; Prescott et al., 1978; George et al., 1982), few havebeen demonstrated in feed to be effective in the treatmentof NE once clinical signs and intestinal lesions have be-come apparent in affected birds (Hamdy et al., 1983a,b).The macrolide antibiotic tylosin has been shown tohave in vitro activity against C. perfringens (Stutz and2001 Poultry Science Association, Inc.Received for publication December 11, 2000.Accepted for publication June 15, 2001.1To whom correspondence should be addressed: email@example.com birds. Mean NE lesion scores on Day 17were signiﬁcantly reduced (P < 0.05) by all levels of tylosintreatment compared to those of unmedicated birds, de-creasing linearly from 2.66 at 0 ppm to 0.38 at 100 ppmand 0 at higher doses. Tylosin at all levels provided im-provement in Day 29 body weight, average daily gain,feed to gain ratio, and average daily feed intake comparedto unmedicated birds. The results of this study provideevidence that tylosin phosphate, when administered infeed, is effective in the treatment of clinical outbreaks ofNE in broiler chickens and suggest that the optimal dosefor this purpose is 100 ppm.Lawton, 1984; Kondo, 1988; Watkins et al., 1997) andto reduce the incidence of mortality associated with C.perfringens enterotoxaemia in an intraduodenal C. per-fringens inoculation model (Vissiennon et al., 2000). Theobjective of this study was to evaluate the efﬁcacy oftylosin phosphate administered in the feed for the treat-ment of NE in broiler chickens during an induced clinicaloutbreak mimicking typical conditions of modern poul-try husbandry.MATERIALS AND METHODSA total of 2,000 1-d-old Cobb × Cobb broiler chicksobtained from a commercial hatchery were used in thisstudy. Birds were vaccinated for Marek’s disease at thehatchery but received no other medications throughoutthe study, other than tylosin phosphate. Replacementswere not made for early mortality. The research facilitywas thoroughly cleaned and disinfected prior to birdplacement. Treatment groups were randomly assigned to40 pens on concrete ﬂoors with new wood shavings forbedding. The area of each pen was 4.2 m2, and eachcontained 25 male and 25 female birds at trial commence-Abbreviation Key: NE = necrotic enteritis.
BRENNAN ET AL.1452ment. Pens were separated by solid plastic barriers ex-tending 30 cm up from ﬂoor level followed by 90 cm ofwelded wire. Lighting program, heating, ventilation, andother management procedures were typical of modernintensive broiler farms in Ontario, Canada. Water wasprovided ad libitum by four nipple-type drinkers per pen.Dry feed was provided ad libitum by one tube-type feederper pen, except for a 12-h period of feed withdrawalimmediately prior to C. perfringens inoculum administra-tion and during the challenge period itself, when inocu-lum-feed mixture was provided in trough-type feeders.All experimental procedures were conducted in accor-dance with the guidelines of the Canadian Council onAnimal Care (1993).A C. perfringens challenge model, based on that devel-oped originally by Prescott et al. (1978), was used to initi-ate an outbreak of NE among the experimental animals.The model, with minor modiﬁcations, has been describedin a number of subsequent publications (Brennan et al.,1996; Skinner and Brennan, 1999). Inoculum was adminis-tered via the feed commencing Day 14 and ending Day16. Birds were fed a commerical wheat-based broilerstarter diet from Days 0 to 21, except for Days 8 to 13when a high protein starter containing 50% ﬁshmeal (arecognized risk factor for naturally occurring outbreaksof NE) (Truscott and Al-Sheikhly, 1977) was fed. A com-mercial broiler grower diet was fed following the termina-tion of the treatment period on Day 22 until trial termina-tion on Day 29.The challenge inoculum contained between 2 × 108and3 × 108cfu C. perfringens/mL at administration. The inocu-lum was prepared from a culture of C. perfringens isolatedfrom a bird displaying severe lesions of NE.Birds that were moribund and unable to reach food orwater were culled and euthanized by cervical dislocation.All dead or culled birds were necropsied to determinethe cause of morbidity or death. A gross pathologic diag-nosis of NE was based on the presence of intestinal lesionstypical of naturally occurring and experimentally pro-duced NE. These lesions consisted of focal to conﬂuentareas of bland mucosal necrosis of the small intestine,often forming a pseudomembrane with no gross evidenceof inﬂammatory reaction or hemorrhage. All dead orculled birds were also scored for intestinal lesions of NEas follows: 0 = no gross lesions; 1 = thin walled or friable;2 = focal necrosis or ulceration; 3 = large patches of necro-sis; 4 = severe extensive necrosis (Prescott et al., 1978). Inaddition, on Day 15 one male bird per pen was euthanizedwith inhaled carbon dioxide gas, subjected to necropsy,and scored for gross lesions of NE and coccidiosis, thelatter according to criteria established for ﬂoor-pen stud-ies in chickens (Johnson and Reid, 1970).Criteria for declaring an NE outbreak were establishedprior to commencement of the study and consisted of thefollowing: 1) observation of clinical signs in the ﬂockconsistent with naturally occurring and experimentallyproduced outbreaks of NE (consisting of some combina-tion of depression, huddling, reduced feed intake, andchanges in fecal consistency), and 2) at least 30% of birdsrandomly selected on Day 15 for euthanizing having grossintestinal lesions typical of NE as deﬁned above, or 3) atleast 30% of randomly selected and euthanized birds andspontaneous mortalities postinoculation (and prior to ini-tiation of treatments) combined having gross intestinallesions of NE.On Day 15 an outbreak of NE was declared based onthe following criteria: 1) the ﬂock had clinical signs (asdescribed above) consistent with an outbreak of NE, 2)36/40 (90%) randomly selected and euthazied birds hadgross intestinal lesions consistent with NE and no grossevidence of coccidiosis, and 3) 5/6 (83%) birds founddead on Day 15 had gross intestinal lesions consistentwith NE.Following identiﬁcation of the outbreak on Day 15,treatments consisting of ﬁve levels of tylosin phosphate(0, 50, 100, 200, or 300 ppm), administered in the feed forseven consecutive days (Days 15 to 22), were randomlyallocated at the pen level in a randomized complete blockdesign. Each of the eight blocks consisted of ﬁve adjacentpens within the trial facility. The investigators and techni-cal staff were not informed of the treatment assignmentuntil the termination of the study.Four birds were randomly selected from each pen (twoof each sex, total 160 birds) on each of d 17 and 22,euthanized, and scored for gross lesions of NE and coccid-iosis, as described above. Birds that died or were euthan-ized for humane purposes postchallenge (Day 14) weresubmitted for detection of C. perfringens. Gram-stainedsmears of intestine were examined for the presence oftypical organisms; samples of the same tissues were cul-tured anaerobically on blood agar at 37 C and examinedafter 20 h for the presence of colonies exhibiting typicalgrowth characteristics (smooth, entire, and with a doublezone of hemolysis) (Ficken and Wages, 1997). Birds thatdied or were euthanized for humane purposes after Day14 were classiﬁed as positive for NE if they had grosspathologic and microbiologic ﬁndings consistent withthat diagnosis.Body weights were measured on a pen basis on Days0 and 29. Feed consumption was measured on a pen basistwice daily during the 3-d challenge and over the entirestudy. One sample of each feed was analyzed for mois-ture, crude protein, ash, fat, calcium, and phosphorus. ATABLE 1. Mortality and lesion scores in a study of the efﬁcacy oftylosin phosphate in the treatment of necroticenteritis (NE) in broiler chickensTylosin phosphate NE mortality, Mean NE lesion Mean NE lesion(ppm) Days 15–29 (%) score, Days 17 score, Days 220 3.25a2.66a0.63a50 0b1.41b0.32a,b100 0.25b0.38c0.006a,b,1200 0.5b0d0.006a,b,1300 0.25b0d0ba–cMeans within a given column with no common superscript are sig-niﬁcantly different (P < 0.05).1Day 22 mean NE lesions scores at 100 and 200 ppm are not signiﬁcantlydifferent from 0 ppm (P = 0.08).
EFFICACY OF TYLOSIN FOR TREATMENT OF NECROTIC ENTERITIS 1453TABLE 2. Growth performance measures in a study of the efﬁcacy of tylosin phosphatein the treatment of necrotic enteritis (NE) in broiler chickensAverage dailyTylosin phosphate Body weight, Average daily gain1feed intake1(ppm) Day 29 (kg) (kg/bird/day) Feed:gain1(kg/bird/day)0 1.020a0.032a1.740a0.055a50 1.141b0.037b1.642b0.060b100 1.160b0.037b1.629b,c0.061b200 1.164b0.037b1.607c0.059b300 1.157b0.037b1.630b,c0.060ba,bMeans within a given column with no common superscript are signiﬁcantly different (P < 0.05).1Adjusted for mortalities and removals due to morbidity.sample of each feed was also analyzed for tylosin contentusing HPLC. Permitted analytical variation of drug con-tent for all medicated feeds was ± 25% of the calculateddrug content.Statistical analyses were conducted using the SAS Sys-tem (SAS Institute, 1997). Necrotic enteritis pen mortalitydata were analyzed by a series of pairwise treatmentcomparisons using an exact P-value for the median test(SAS Proc NPAR1WAY). For the purposes of this analysis,spontaneous mortalities and moribund birds that wereeuthanized for humane purposes and that had a ﬁnaldiagnosis of NE were treated identically. The Mann-Whit-ney-Wilcoxon test (exact P-value from SAS Proc StatXact)was used to conduct pairwise comparisons by treatmentof mean pen lesion scores. Analysis of variance was usedto test the effect of treatment on mean ﬁnal body weight,average daily gain, ratio of feed to gain, and average dailyfeed intake. The pen was considered the experimentalunit for all analyses.RESULTSAll feeds were within tolerance for analyzed drug andnutrient content. The addition of tylosin phosphate to thefeed signiﬁcantly reduced mortality (P < 0.05) due to NEat all dose levels when compared to unmedicated birds(Table 1).Mean NE lesion scores on Day 17 were signiﬁcantlyreduced (P < 0.05) by all levels of tylosin compared tounmedicated birds, decreasing linearly from 2.66 at 0 ppmto 0.38 at 100 ppm and 0 at higher doses (Table 1).On Day 22, mean NE lesion scores were essentiallyequal to or lower than those observed at correspondingdose levels on Day 17. The mean NE lesion score observedon Day 22 was signiﬁcantly lower (P < 0.05) for birdsadministered tylosin at 300 ppm compared to unmedi-cated birds (Table 1). Birds administered tylosin at 100and 200 ppm had mean NE scores that were lower thanunmedicated birds but were not signiﬁcantly different (P= 0.08). Coccidiosis lesion scores were 0 on Days 17 and22 for all euthanized birds.Tylosin at all levels provided improvement in Day 29body weight, average daily gain, feed to gain ratio, andaverage daily feed intake compared to unmedicated birds(Table 2).DISCUSSIONThe results of this study demonstrate that tylosin phos-phate is effective in reducing morbidity, mortality, andsuppression of growth and feed efﬁciency associated withNE in broiler chickens, when administered in the feedafter onset of an outbreak.These ﬁndings are consistent with previous studies ofthe activity of tylosin against C. perfringens in vitro (Stutzand Lawton, 1984; Kondo, 1988; Watkins et al., 1997) andsupport the ﬁnding of Vissiennon et al. (2000) that tylosinis effective in reducing mortality associated with thisdisease.Signiﬁcantly reduced mean lesion scores among medi-cated birds on Days 17 and 22 indicate that, in addition toreducing mortality, administration of tylosin signiﬁcantlyreduced the clinical impact of challenge among survivingbirds. This result suggests that the positive effect of medi-cation on growth parameters (Table 2) was related tocontrol of NE and not simply a result of a growth pro-moter effect.All dose levels of tylosin signiﬁcantly reduced mortal-ity and mean lesion scores on Day 17 and improvedgrowth parameters. For Day 17, birds receiving 100 ppmhad signiﬁcantly reduced mean lesion scores comparedto those receiving 50 ppm. Although on Day 17, the birdsreceiving 200 ppm dose had signiﬁcantly lower meanlesion scores compared to those receiving 100 ppm, themagnitude of the effect is not considered by the authorsto be of clinical relevance.The results of this study thus provide evidence thattylosin phosphate, when administered in feed, is effectivein the treatment of clinical outbreaks of NE in broilerchickens and suggest that the optimal dose for this pur-pose is 100 ppm.REFERENCESBrennan, J. J., J. Radu, D. A. Barnum, D. Carrier, J. Kelly, andB. Kilmer, 1996. Efﬁcacy of zinc bacitracin and bacitracinmethylene disalicyclate for prevention of necrotic enteritisin broiler chickens. Poultry Sci. 75(Suppl. 1):283. (Abstr.).Canadian Council on Animal Care, 1993. Guide to the Care andUse of Experimental Animals. Vol. 1. 2nd ed. E. D. Olfert,B. M. Cross, and A. A. McWilliam, ed. CCAC Ottawa,ON, Canada.
BRENNAN ET AL.1454Ficken, M. D., and D. P. Wages, 1997. Necrotic enteritis. Pages261–264 in: Diseases of Poultry. 10th ed. B. W. Calnek, ed.Iowa State University Press, Ames, IA.George, B. A., C. L. Quarles, and D. J. Fagerberg, 1982. Virgin-iamycin effects on controlling necrotic enteritis infection inchickens. Poultry Sci. 61:447–450.Hamdy, A. H., R. W. Thomas, D. D. Kratzer, and R. B. Davis,1983a. Lincomycin dose response for treatment of necroticenteritis in broilers. Poultry Sci. 62:585–588.Hamdy, A. H., R. W. Thomas, R. J. Yancey, and R. B. Davis,1983b. Therapeutic effect of optimal lincomycin concentra-tion in drinking water on necrotic enteritis in broilers. PoultrySci. 62:589–591.Johnson, J. K., and W. M. Reid., 1970. Anticoccidial drugs: Lesionscoring techniques in battery and ﬂoor-pen experiments withchickens. Exp. Parasitol. 28:30–36.Kondo, F., 1988. In vitro lecithinase activity and sensitivity to22 antimicrobial agents on Clostridium perfringens isolatedfrom necrotic enteritis of broiler chickens. Res. Vet. Sci.45:337–340.Long, J. R., and R. B. Truscott, 1976. Necrotic enteritis in broilerchickens. III. Reproduction of the disease. Can. J. Comp. Med.40:53–59.Prescott, J. F., R. Sivendra, and D. A. Barnum, 1978. The use ofbacitracin in the prevention and treatment of experimentally-induced necrotic enteritis in the chicken. Can. Vet. J.19:181–183.SAS Institute Inc., 1997. SAS/STAT Software: Changes andEnhancements Through Release 6.12, SAS Institute Inc.,Cary, NC.Skinner, J,. and J. Brennan, 1999. Efﬁcacy of bacitracin methylenedisalicyclate fed in combination with narasin for preventionof necrotic enteritis. Poultry Sci. 78(Suppl. 1):129. (Abstr.).Stutz, M. W., and G. C. Lawton, 1984. Effects on diet and antimi-crobials on growth, feed efﬁciency, intestinal Clostridium per-fringens, and ileal weight of broiler chicks. Poultry Sci.63:2036–2042.Truscott, R. B., and F. Al-Sheikhly, 1977. Reproduction andtreatment of necrotic enteritis in broilers. Am. J. Vet. Res.38:857–861.Vissiennon, Th., H. Kro¨ger, T. Ko¨hler, and R. Kliche, 2000. Effectof avilamycin, tylosin and ionophore anticoccidials on Clos-tridium perfringens enterotoxaemia in chickens. Berl. Munch.Tiera¨rztl. Wschr. 113:9–13.Watkins, K. L., T. R. Shryock, R. N. Dearth, and Y. M. Saif, 1997.In vitro antimicrobial susceptibility of Clostridium perfringensfrom commercial turkey and broiler chicken origin. Vet. Mi-crobiol. 54:195–200.