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Research on the Probable Cause of an Outbreak of Field Rickets in Turkeys1 W. E. HUFF,*,2 G. R. HUFF,* F. D. CLARK,† P. A. MOORE, JR.,* N. C. RATH,* J. M. BALOG,* D. M. BARNES,† G. F. ERF,† and K. W. BEERS† *USDA, Agricultural Research Service, Poultry Production and Product Safety Research Unit, Poultry Science Center and †Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 ABSTRACT An outbreak of ﬁeld rickets in turkeys chemistries. In the third study, there were ﬁve dietary prompted studies on the cause. In Experiment 1, there treatments with two replicate pens of 25 poults per treat- were four treatments with two replicate pens of 10 poults ment. The treatments consisted of poults fed newly manu- per pen. The treatments consisted of poults fed newly factured feed; new feed mixed with 25, 50, or 75% suspect manufactured feed (control), poults fed a diet containing feed; or 100% suspect feed. Body weights of poults fed control feed and 5% clean litter, poults fed control feed 100% suspect feed were decreased at 2, 3, and 4 wk as and 5% litter from the pens of affected poults, and poults was the relative weight of the liver, pancreas, and bursa challenged with an intestinal homogenate by gavage. of Fabricius. The relative weight of the kidney increased. Field rickets did not develop with these treatments. The Lameness, a decrease in bone ash, and changes in hema- feed was suspect, and, in Experiment 2, poults were either tology and blood chemistry were observed in the poults fed the suspect feed or newly manufactured feed. There consuming 100% suspect feed. These data demonstrate were four replicate pens of 25 poults per pen. Poults fed that feed from the original outbreak could induce ﬁeld the suspect feed had a decrease (P ≤ 0.05) in BW at 1, 2, rickets and was toxic. Because the feed contained ade- 3, and 4 wk of age; an increase in the relative weight of quate vitamin D, calcium, and phosphorus, the cause of the liver, pancreas, kidney, and bursa of Fabricius; and this outbreak of ﬁeld rickets is thought to be a toxic feed a decrease in bone ash. There were changes in clinical contaminant affecting bone development. (Key words: ﬁeld rickets, turkeys, poults, feed) 1999 Poultry Science 78:1699–1702 INTRODUCTION multiple causes of ﬁeld rickets seem reasonable. The ob- jective of these studies was to determine the probable We experienced a severe outbreak of rickets in our cause of this outbreak of ﬁeld rickets. facility with over 90% of the poults affected at 2 wk of age. This outbreak was characterized by lameness, soft MATERIALS AND METHODS and rubbery bones, and beaded ribs, although more than adequate levels of calcium, phosphorus, and vitamin D The original outbreak of ﬁeld rickets occurred in an were present in the diet. Field rickets has been deﬁned experiment with 240 male and 240 female turkey poults as a rachitic condition that occurs even though the diets (Nicholas 600s) housed in pine litter ﬂoor pens with 20 are adequate in calcium, phosphorus, and vitamin D turkeys per pen. Over 90% of these poults became lame (Hurwitz et al., 1973). Field rickets occurs in all turkey- at 2 wk of age. Upon necropsy, the ribs were beaded and producing regions and has been studied by a number of the bones were found to be soft and rubbery; rickets researchers (Hurwitz et al., 1973; Walser et al., 1980; Olson was diagnosed. The feed had been formulated to meet or et al., 1981; Riddell, 1983; Bar et al. 1987). The etiology of exceed NRC recommendations (NRC, 1994). The calcium ﬁeld rickets remains unknown, and, given the complexity and phosphorus levels of the diet were determined with of mineral and vitamin D utilization and metabolism, an inductively coupled argon emission spectrophotome- ter3 following the manufacturer’s procedures, and the levels were found to be consistent with the formulation (1.22 and 0.77%, respectively). The vitamin D level in the Received for publication April 19, 1999. diet was also found to be as formulated (2,204 IU) using Accepted for publication August 11, 1999. 1 Mention of a trade name, proprietary product, or speciﬁc equipment the method of Singh and Bradbury (1988). does not constitute a guarantee or warranty by the USDA and does This outbreak of ﬁeld rickets prompted three studies not imply its approval to the exclusion of other products that may to determine the probable cause. In Experiment 1, there be suitable. 2 Correspondence should be addressed to: email@example.com were four treatments with two replicate pens of 10 male 3 Model D, Spectro Analytical Instruments, Inc., Fitchburg, MA 01420. poults (Nicholas 600). The treatments consisted of a con- 1699 1700 HUFF ET AL. TABLE 1. The effect of newly manufactured feed and suspect feed on BW,1 the relative weight of the liver1 and kidney,1 bone strength,2,3 bone ash,2 and serum levels of calcium2 and phosphorus2 Treatment BW at 28 d Liver Kidney Strength Ash Ca P (% BW) (N/m × 10 ) 2 6 (%) (mg/dL) New feed 1,278 ± 14a 2.39 ± 0.04b 0.41 ± 0.01b 28.6 ± 1.1a 47.3 ± 0.4a 10.6 ± 0.2a 7.6 ± 0.2a Suspect feed 544 ± 10b 2.84 ± 0.03a 0.46 ± 0.01a 9.0 ± 0.5b 42.4 ± 0.6b 8.9 ± 0.4b 5.5 ± 0.5b Values within a column with different superscripts differ (P ≤ 0.05). a,b 1 Values represent the means ± SEM of four replicate pens of 25 poults per pen. 2 Values represent the means ± SEM of four replicate pens of ﬁve poults per pen. 3 Values for bone strength have been corrected for differences in tibia diameter as described by Huff et al. (1980). trol (newly manufactured feed), the new feed containing All data presented as percentages were arc sine trans- 5% litter taken from the pens of the affected poults, the formed prior to statistical analysis. Signiﬁcant differences new feed containing 5% fresh litter, and challenge with among treatments were separated using Duncan’s multi- 1 mL of an intestinal homogenate made from the affected ple range test (Duncan, 1955). All statements of signiﬁ- poults delivered by gavage at 1 d of age. The birds were cance are based on the probability level of 0.05. housed in batteries, and feed and water were available for ad libitum consumption from 1 d to 4 wk of age. RESULTS In Experiment 2, there were two treatments of either the feed consumed by the poults that developed ﬁeld In the original outbreak of ﬁeld rickets, over 90% of the rickets (suspect feed) or newly manufactured feed. There poults were affected, characterized by lameness, beaded were four pens of 25 male poults (Nicholas 600) in each ribs, and rubbery bones, despite an adequate amount of of the two treatments; again, feed and water were avail- calcium, phosphorus, and vitamin D in the diets. There able for ad libitum consumption from 1 d to 4 wk of age. were no signs of lameness or rickets in the poults in When the poults were 4 wk of age, ﬁve poults per pen Experiment 1. The effects of the treatments in Experiment were bled by cardiac puncture, and various hematological 2 on BW, the relative weights of the liver and kidney, evaluations were performed using a Model 3500 Cell- bone strength, bone ash, and serum levels of calcium Dyn hematology analyzer4 following the manufacturer’s and phosphorus are presented in Table 1. There was a recommendations. Clinical chemistry analysis of serum signiﬁcant decrease in the BW of the poults consuming was performed using an automated clinical chemistry the suspect feed. There was a slight yet signiﬁcant increase analyzer5 following manufacturer’s procedures. The in the relative weight of the liver and kidney in the poults poults were killed by cervical dislocation, and various consuming the suspect feed. Bone strength was decreased organs were excised and weighed. The left tibia was re- threefold in the birds fed the suspect feed. Bone ash and moved from ﬁve poults per pen, and breaking strength serum levels of calcium and phosphorus were also sig- was determined using an Instron Model 4502 Shear Press6 niﬁcantly decreased in the poults consuming the sus- as described by Huff et al. (1980). The bones for bone ash pect feed. analysis were fat-extracted in a Soxhlet extractor for 16 In Experiment 3, the suspect feed was blended with h with ethanol followed by 16 h with ethyl ether and then newly manufactured feed, and the effects of these treat- ashed at 750 C for 17 h. ments on BW; the relative weights of the liver and kidney; The treatments in Experiment 3 consisted of control bone strength; bone ash; and the serum levels of calcium, feed (newly manufactured) and control feed containing phosphorus, and alkaline phosphatase are presented in 25, 50, 75, and 100% suspect feed. There were two pens Table 2. There was a signiﬁcant increase in BW with the of 25 male poults (Nicholas 600) in each of the treatments, diet containing 25% suspect feed, and a signiﬁcant and and feed and water were available for ad libitum consump- substantial decrease in BW with the 100% suspect feed tion. The poults were bled and necopsied when they were diet. The relative weight of the liver was signiﬁcantly 25 d of age. All other procedures and evaluations were decreased with the 100% suspect feed diet, and the rela- as described for Experiment 2. At the start of Experiment tive weight of the kidney was signiﬁcantly increased with 3, the suspect feed was 8 wk old. the 50 and 100% suspect feed diet. Bone strength was A t-test was used to evaluate the data in Experiment signiﬁcantly decreased with the 75 and 100% suspect feed 2. The data in Experiment 3 were anayzed by ANOVA diets, and bone ash was signiﬁcantly decreased only when (Snedecor and Cochran, 1967) using the General Linear the 100% suspect feed diet was fed. There was a signiﬁcant Models procedure of SAS software (SAS Institute, 1998). decrease in serum calcium levels with the 100% suspect feed diet. The serum phosphorus levels were inconsistent with a signiﬁcant increase in phosphorus levels in the 4 75% suspect feed diet, and there was a signiﬁcant increase Abbott Diagnostics, Abbott Park, IL 60064. 5 Express Plus, Ciba-Corning Diagnostics Corp., Medﬁeld, MA 02052. in the serum activity of alkaline phosphatase with the 6 Instron Corp., Canton, MA 02021. 100% suspect feed diet. RESEARCH NOTE 1701 1 1 TABLE 2. The effect of diets blended with suspect feed on BW; the relative weight of the liver and kidney; bone strength; bone ash;2 1 2 and the serum levels of calcium,2 phosphorus,2 and alkaline phosphatase (Alk. Phos.)2 Treatment BW at 25 d Liver Kidney Strength Ash Ca P Alk. Phos. (% BW) (N/m × 10 ) 2 6 (%) (mg/dL) (IU/L) Control 643 ± 11bc 2.78 ± 0.05a 0.50 ± 0.01b 15.2 ± 0.9a 46.6 ± 0.5ab 11.5 ± 0.3a 6.8 ± 0.5bc 4,676 ± 300b 25%3 705 ± 14a 2.70 ± 0.04a 0.50 ± 0.01b 18.2 ± 0.8a 48.1 ± 0.4a 11.1 ± 0.2a 8.2 ± 0.2ab 4,670 ± 158b 50% 684 ± 11ab 2.86 ± 0.06a 0.55 ± 0.01a 15.9 ± 0.9a 47.1 ± 0.6ab 10.8 ± 0.2a 7.4 ± 0.3abc 4,355 ± 323b 75% 618 ± 19c 2.76 ± 0.04a 0.53 ± 0.01ab 9.5 ± 1.7b 45.7 ± 0.8b 11.8 ± 0.4a 8.6 ± 0.5a 4,972 ± 792b 100% 264 ± 9d 2.48 ± 0.05b 0.56 ± 0.01a 2.8 ± 0.3c 36.6 ± 1.2c 6.3 ± 0.8b 6.0 ± 0.7c 13,099 ± 1,079a Values within a column with no common superscript differ (P ≤ 0.05). a–d 1 Values represent the means ± SEM of two replicate pens of 25 poults per pen. 2 Values represent the means ± SEM of two replicate pens of ﬁve poults per pen. 3 Percentage of suspect feed in diet. The effect of these treatments on red blood cell counts, toxic, characterized by a decrease in BW, affecting the serum levels of cholesterol, triglycerides, uric acid, and relative weights of the liver and kidney, as well as changes protein are presented in Table 3. Serum levels of choles- in hematology and clinical chemistries. These data clearly terol and triglycerides were signiﬁcantly decreased, and indicate that the probable cause of this outbreak of ﬁeld red blood cell counts and the serum levels of uric acid rickets was associated with the feed, even though the and protein were signiﬁcantly increased with the 100% feed had adequate minerals and vitamin D. The effect of suspect feed diet. this feed on the relative weights of the liver and kidney suggests that vitamin D metabolism in the liver and kid- DISCUSSION ney could have been affected. In the absence of the isolation and characterization of Considering the complexity of bone formation, it is this toxic component of the feed, mycotoxin contamina- reasonable to suspect that there may be multiple causes tion of the feed would seem to be a reasonable hypothesis. of ﬁeld rickets. Infectious agents could disrupt the utiliza- Several mycotoxins have been shown to affect nutrient tion of minerals and vitamin D through the pathology availability (Hamilton et al., 1974), as well as bone forma- caused in the liver, kidney, or intestinal tract. Although tion (Huff et al., 1980). In addition, mycotoxins produced most of the work on ﬁeld rickets discounts infectious by species of the genus Fusarium have been shown to agents being involved (Walser et al., 1980; Olson et al., induce a rachitic-type syndrome in broilers (Gedek et al., 1981; Riddell, 1983), others suggest that infectious agents 1978), and fusarochromanone can cause tibial dyschon- may be involved (Vertommen et al., 1980a,b). In Experi- droplasia (Chu et al., 1988; Wu et al., 1991, 1993; Chu et ment 1, we could not induce ﬁeld rickets either by incor- al., 1995). However, to the authors’ knowledge no speciﬁc porating litter from the pens of affected poults into newly mycotoxin has been shown to cause ﬁeld rickets, and any manufactured feed or by challenging turkeys at 1 d with number of other feed contaminants must be considered an intestinal homogenate from affected poults, which sug- as suspect. Regardless of the identity of the toxic principal gests that the ﬁeld rickets we observed was not caused in the feed, it seems that the best approach to the treat- by an infectious agent. ment of ﬁeld rickets would be to replace or blend the Although lameness was not observed in Experiment 2, feed. Massive vitamin D therapy in outbreaks of ﬁeld the poults in both Experiments 2 and 3 had rickets, which rickets has not always been successful (Bar et al., 1987; was characterized by a decrease in bone strength, bone Riddell, 1983). If the cause of ﬁeld rickets is a yet-to-be ash, and serum levels of calcium and phosphorus in Ex- identiﬁed toxin that affects vitamin D metabolism, the periment 2 and a decrease in calcium in Experiment 3. toxic affects might be overwhelmed with massive levels In addition to causing rickets, the suspect feed was also of vitamin D. However, it would seem more reasonable TABLE 3. The effect of diets blended with suspect feed on red blood cell counts (RBC) and the serum levels of cholesterol, triglycerides, uric acid, and protein1 Treatment RBC Cholesterol Triglycerides Uric Acid Protein (mm 3 × 10 ) 6 (mg/dL) (g/dL) Control 2.5 ± 0.1b 105 ± 6a 92 ± 12a 7.1 ± 0.8b 2.9 ± 0.1b 25%2 2.5 ± 0.1b 103 ± 5a 71 ± 6ab 8.1 ± 0.8b 3.0 ± 0.1b 50% 2.5 ± 0.1b 91 ± 5ab 65 ± 6ab 8.4 ± 0.8b 3.0 ± 0.1b 75% 2.5 ± 0.1b 100 ± 11a 77 ± 10a 11.1 ± 1.0b 3.3 ± 0.2b 100% 2.9 ± 0.1a 73 ± 9b 44 ± 10b 30.4 ± 0.8a 4.1 ± 0.3a Values within a column with no common superscript differ (P ≤ 0.05). a,b 1 Values represent the means ± SEM of two replicate pens of ﬁve poults per pen. 3 Percentage of suspect feed in diet. 1702 HUFF ET AL. to treat ﬁeld rickets with the vitamin D metabolites of Huff, W. E., J. A. Doerr, P. B. Hamilton, D. D. Hamann, R. E. either 25-hydroxy cholecalciferol or 1, 25 di-hydroxy cho- Peterson, and A. Ciegler, 1980. Evaluation of bone strength during aﬂatoxicosis and ochratoxicosis. Appl. Environ. Mi- lecalciferol. This approach is supported by the work of crobiol. 40:102–107. Hurwitz and Bar (1981), which showed that 25-hydroxy Hurwitz, S., and A. Bar, 1981. Calcium, phosphorus, and vitamin cholecalciferol was more effective at treating ﬁeld rickets D deﬁciencies in young turkeys: Diagnosis and treatment. in turkeys than vitamin D. Minnesota Turkey Research. Univ. Minn. Agric. Exp. Stn. The complexity of calcium, phosphorus, and vitamin Rep. 179:33–41. D availability and utilization in bone development would Hurwitz, S., A. Bar, and A. Meshorer, 1973. Field rickets in turkey poults: Plasma and bone chemistry, bone histology, suggest that ﬁeld rickets could be caused by a number intestinal, calcium-binding protein. Poultry Sci. 52:1370– of factors. Although the feed was determined to have 1374. adequate levels of calcium, phosphorus, and vitamin D, National Research Council, 1994. Nutrient Requirements of it was shown to be the probable cause of ﬁeld rickets in Poultry. 9th rev. ed. National Academy Press, Washing- these studies. The fact that the feed was also toxic suggests ton, DC. that a contaminating toxin, possibly a mycotoxin, might Olson, W. G., H. E. Dziuk, M. M. Walser, G. F. Hanlon, P. E. Waibel, J. B. Stevens, and N. A. Jorgensen, 1981. Field rickets have been responsible for this outbreak of ﬁeld rickets. in turkey poults: Biochemical ﬁndings. Avian Dis. 25:550– 554. ACKNOWLEDGMENTS Riddell, C., 1983. Rickets in turkey poults. Avian Dis. 27:430–441. SAS/STAT User’s Guide: 1998 Edition. SAS Institute Inc., The authors gratefully acknowledge the excellent tech- Cary, NC. nical assistance of Scott Zornes, Dana Bassi, David Hor- Singh, U., and J. H. Bradbury, 1988. HPLC determination of lick, Sonia Tsai, and Chris Taylor. vitamin A and vitamin D2 in south paciﬁc root crops. J. Sci. Food Agric. 45:87–94. REFERENCES Snedecor, G. W., and W. G. 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