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Flowers of Sulphur and Charcoal in the Prevention of Experimentally Produced Coccidiosis*
Flowers of Sulphur and Charcoal in the Prevention of Experimentally Produced Coccidiosis* O.
E.
GOFF
Louisiana State University, Agricultural Experiment Station, Baton Rouge, Louisiana (Received for publication May 12, 1941)
V
AN ES and Olney (1940) set forth the opinion of many poultrymen when they stated that, although coccidiosis might be controlled to a bearable extent by sanitation and management, success in its complete elimination by these measures was difficult. Herrick and Holmes (1936) and Dickinson and Scofield (1939) reported that flowers of sulphur aided in the prevention of Eimeria tenella infection. In a preliminary report Goff and Upp (1940a) found that the coccidiosis preventive properties of sulphur were enhanced when 5 percent charcoal was included in a ration containing 5 percent of any one of the four grades of sulphur studied. Some of the effects of sulphur upon the chicks have been reported by Holmes, Deobald, and Herrick (1938), Dickinson and Schofield (1939), and Goff and Upp (1940b). Additional data regarding the coccidiosis preventive properties of sulphur and charcoal have been collected and a part of these are presented in this paper. METHODS AND MATERIALS
The technic for preparing a suspension of coccidian oocysts for inoculation purposes as reported by Goff and Upp (1940a) has been simplified and consequently made more useable and rapid. Oocysts of Eimeria tenella were obtained by killing culture in* Published by approval of the director of the Louisiana Agricultural Experiment Station. This study was partially supported through a fellowship provided by the Freeport Sulphur Company.
fected chicks on the eighth or ninth day following inoculation. The ceca were removed, split, placed in Petri dishes, and moistened with a 2.5 percent solution of potassium dichromate. Cecal cores were crushed with a pair of forceps to aid aeration and sporulation. Few oocysts appeared to be present in the ceca at the end of the sixth day following inoculation; great numbers were present on the eighth day. Twelve days after inoculation many cecal cores had been passed and the number of oocysts present in the cecal content had greatly decreased; of those that were present the percent sporulation was usually low. Sporulation began by the eighteenth hour and continued through 48 hours. However, sporulation was practically complete after 24 hours, when the material was maintained at a temperature of approximately 75°F. After sporulation the best samples were combined, all ceca were scraped, and the material thus obtained ground through a 729-mesh per square inch wire sieve. Oocysts were concentrated from this material by repeated washing and straining and finally by sedimentation. Oocyst counts and inoculations of measured quantities of this inoculum were made in the manner described by Goff and Upp (1940a). This procedure has several advantages over the one previously presented in that it saves time, less expensive equipment is needed, oocysts are more numerous in cecal material
[23]
24
O. E. GOFF
than in droppings, oocysts have consistently sporulated better than those in cecal droppings, it is more pleasant to work with fresh cecal material, and the danger of contamination with other species of coccidia is greatly decreased. Flowers of sulphur and No. 10 hardwood oak charcoal were used in these trials. The charcoal is commonly referred to by the poultrymen as chick-size poultry charcoal. The adsorptive properties of this product are less than the finely divided product ordinarily used in biological laboratories for adsorption of vitamins, hormones, enzymes, and others, and appears to be a different product than the adsorbing charcoal used by Almquist and Zander (1940). All chicks were two weeks of age or older before they received any elemental sulphur. As indicated in Tables, 1, 2, and 3 the various lots were fed rations containing 2 or S percent flowers of sulphur with and without 5 percent No. 10 oakwood charcoal. One lot received S percent charcoal but no sulphur, while the control lots received the basal ration throughout with neither sulphur nor charcoal. All chicks were weighed individually every two weeks. All chicks were maintained in electrically heated battery brooders under conditions unfavorable to natural infection. The chicks used in June and July of 1939 were Single Comb White Leghorns and White Plymouth Rocks; those used in the later trials were White Plymouth Rocks. The basal ration used was the same as reported by Goff and Upp (1940a). Prior to the first inoculaton the cecal droppings of each chick were examined for oocysts, and in every case the findings were negative. At the time of hemorrhage the chicks were confined in individual chick batteries. As it is not known how many coccidian oocysts chicks might ingest under conditions favoring natural infection, it was deemed desirable to inoculate with large numbers
of sporulated oocysts to more rigidly test the coccidiosis preventive properties of flowers of sulphur. Thus, from 400,000 to almost 2,000,000 sporulated oocysts were administered each chick as indicated in Tables 1 and 2. RESULTS AND DISCUSSION
Data presented in Table 1 revealed that the chicks receiving flowers of sulphur (with or without charcoal) prior to inoculation with measured doses of E. tenella suffered less mortality from artificially induced coccidiosis than did chicks fed rations containing no sulphur. Highly consistent results were secured in each of the six trials. Chisquare tests revealed that no significant differences existed in coccidiosis mortality between the inoculated chicks receiving the basal ration and those receiving the ration containing 5 percent charcoal. All data involving chi-square were corrected for continuity by Yates' formula as suggested by Snedecor (1937). The chicks inoculated in each group and fed 2 percent flowers of sulphur plus S percent charcoal gave a greater percent survival than chicks receiving 2 percent sulphur without charcoal. This difference remained highly significant after correcting for continuity. Rations containing 2 percent sulphur proved to be inferior, verified statistically, to all other sulphur-containing rations in preventing coccidiosis mortality. When 5 percent charcoal was added to a ration containing 2 percent sulphur the number of chicks that survived the disease was essentially the same as those fed a ration containing 5 percent sulphur; chi-square tests revealed no significant difference. The appearance of the inoculated individuals and the degree of hemorrhage were quite similar for those chicks receiving 2 percent flowers of sulphur plus 5 percent charcoal or 5 percent sulphur without charcoal.
5
5% charcoal, no sulphur
5 5 5
4 5 1 1
4 5
3 5
1
Surv.
5
5 5
5 5
5
Inoc.
0
4 4
2 3
0
Surv.
No. chicks
6 weeks 8 weeks 1,980,000
7/1/39
10
10 10
10 10
10
2 10
10 10
10 10
6 7 8 10
10
0
Surv. Inoc. 10 10 10 10 10 10
0 6 10 10 10 0
Surv. Inoc. 45 45 45 45 45 45
0 7 8 10 10 1
2
X
3 5
X 2
Inoc.
Same controls used for D and E
X
5% charcoal, no sulphur
Group B chicks not included in totals.
9 9
9 10
5%|flowers of sulphur 5% flowers of sulphur plus 5% charcoal. X
6 7
42
Surv.
6 10 10 9
Surv. Inoc.
Surv
Inoc.
Surv.
No. chicks
12/14/39 B 2 weeks1st inoc. 8 weeks 840,000
Surv. 4 18 24 21 29 3
Reinoc. 15 18 27 29 34 3
Total chicks
1
100.0
72.4 85.3
100.0
16.0
Percent survival
11.1
88.9 97.8
62.2 84.4
6.7
X indicates no chicks available for second inoculation.
7 10
3 5 X
6 7
Inoc.
No. chicks
12/14/39 A 2 weeks 4 weeks 8 weeks 840,000
X 2
Surv.
No. chicks
No. chicks
No. chicks Inoc.
1/13/40 E 3 weeks 4 weeks 12 weeks 880,000
1/13/40 D 3 weeks 4 weeks 12 weeks 880,000
12/28/39 C 2 weeks 4 weeks 10 weeks 840,000
2% flowers of sulphur 2% flowers of sulphur plus 5% charcoal
Control (no sulphur). Never previously infected.
Experimental rations previously fed
Date inoculated Group designation Time off experimental ration Time elapsed between inoculations. Age of chick at second inoculation. Sporulated oocysts per chick
5
40 44
28 38
3
Percent Surv. survival
Total chicks
Surv. Inoc.
No. chicks
No. chicks
No. chicks Inoc.
12/14/39 D 4 weeks 8 weeks 840,000
11/30/39 C 2 weeks 6 weeks 780,000
11/19/39 A 2 weeks 4 weeks 780,000
TABLE 2.—Influence of an initial infection of E. tenella upon subsequent immunity, and carryover effect of sulphur
5 5
5 % flowers of sulphur 5 % flowers of sulphur plus 5% charcoal
5 5
4 5
5
2
5 5 S
Inoc.
No. chicks
No. chicks Surv.
6 weeks 8 weeks 660,000
2 weeks 4 weeks 400,000
Inoc.
7/1/39
6/2/39
2% flowers of sulphur
Experimental ration
Date inoculated Group designation Time on experimental diet before inoculation Age of chicks at time of inoculation Sporulated oocysts per chick
TABLE 1.—Value of flowers of sulphur in combination with poultry charcoal in coccidiosis prevention
o o o
o
O
3
W H
<
K w
I-5
IT 1
>
o o
n
a
>
W
r B C
CO d1
o
w
o
26
0. E. GOFF
After the data were corrected for continuity, differences in survival were not significant for chicks fed rations containing either 2 percent flowers of sulphur plus 5 percent charcoal, 5 percent sulphur, or 5 percent sulphur plus 5 percent charcoal. However, the percent survival appears to favor the chicks fed a ration containing 5 percent sulphur plus 5 percent charcoal. The degree of hemorrhage and the ill-appearance of the infected chicks was considered less severe in the 5 percent sulphur and charcoal group. Hemorrhage was observed usually in the no-sulphur lots prior to its occurrence in lots receiving sulphur. The chicks in the 5 percent sulphur and charcoal lot were inactive for a shorter period of time as a result of the disease than were chicks in the other lots; when disturbed these chicks would begin eating while the others would not. Thus the 5 percent sulphur and charcoal ration appeared to be superior to the other rations in preventing coccidiosis. The above data and observations further substantiate the author's belief that mortality alone is an inadequate measure of the severity of coccidiosis but may be used as one of the measures. Four weeks following the first inoculation all survivors were again inoculated with measured amounts of sporulated coccidian oocysts. The results of the reinoculations are recorded in Table 2. At the time of the second inoculations all chicks had received the basal ration without sulphur or charcoal for at least two weeks. Although a high percentage of the chicks survived this second inoculation, complete immunity was not established as all chicks shed oocysts and suffered slight or heavy hemorrhage which appeared quite similar in the various lots. At the time group A chicks were reinoculated (December 14, 1939), a number of group B chicks that had received identical rations, but reared coccidiosis-free, were
inoculated with the same inoculum and number of sporulated oocysts as recorded in Table 2. All group B chicks and control chicks of group A died of acute coccidiosis, while a majority of the reinoculated ones survived. Thus sulphur failed to exert any coccidiosis preventive properties two weeks after it had been removed from chick ra^ tions. At the time group D chicks were inoculated (December 14, 1939), all group E chicks were inoculated for the first time, after receiving the various rations as given in Table 3 for the preceding 22 hours. All group E chicks were given the same amount of inoculum as group D chicks (Table 1). Five chicks per lot were used except in the lot receiving 2 percent sulphur in which only four chicks were available for inoculation purposes. All chicks that received no flowers of sulphur died from acute coccidiosis. Of the chicks receiving 2 percent sulphur, one survived, two survived when fed 2 percent sulphur plus 5 percent charcoal, four survived when protected with 5 percent sulphur, while all five survived when protected with a ration containing 5 percent sulphur plus S percent charcoal. Additional data have been collected regarding this phase of the problem which will be presented in another paper. Reinoculation of chicks in groups C, D, and E provides additional evidence that increased resistance, immunity, or refractiveness was produced by an initial infection with E. tenella to a subsequent reinoculation. However, age undoubtedly influenced survival in groups D and E. At the time chicks in groups D and E were reinoculated (January 13, 1940), 5 chicks, 18 days of age, and 10 chicks, 28 days old, were inoculated with 440,000 sporulated oocysts from the same inoculum. All of these younger chicks died. These data indicate that resistance to coccidiosis was greater in 12-week-old chicks than in the
Control ration throughout. No sulphur. On 2 % F. of S. @ 2 wks. of age; off at 6 wks. On 2% F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5% F. of S. @ 2 wks.; off at 6 wks. On 5% F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % char. @ 2 wks.; off @ 6 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 2 wks.; off @ 6 wks. On 2 % F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % F. of S. at 2 wks.; off @ 6 wks. On 5 % F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % char, at 2 wks.; off @ 6 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 4 wks.; off @ 8 wks. On 2 % F. of S . + 5 % char. @ 4 wks.; off @ 8 wks. On 5 % F. of S. @ 4 wks.; off @ 8 wks. On 5 % F. of S . + 5 % char. @ 4 wks.; off @ 8 wks. On 5% char. @ 4 wks.; off @ 8 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 4 wks.; off @ 9 wks. On 2 % F. of S . + 5 % char. @ 4 wks.; off @ 9 wks. On 5% F. of S. @ 4 wks.; off @ 9 wks. On 5 % F. of S . + 5 % char. @ 4 wks.; off @ 9 wks. On 5% char. @ 4 wks.; off @ 9 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 8 wks.; off @ 9 wks. On 2% F. of S . + 5 % char. @ 8 wks.; off @ 9 wks. On 5 % F. of S. @ 8 wks.; off @ 9 wks. On 5% F. of S . + 5 % char. @ 8 wks.; off @ 9 wks. On 5 % char. @ 8 wks.; off @ 9 wks.
601A 602A 603A 604A 605A 606A
601B 602B 603B 604B 605B 606B
601C 602C 603C 604C 605C 606C
601D 602D 603D 604D 605D 606D
601E 602E 603E 604E 605E 606E 35(5) 37(4) 38(5) 35(5) 36(5) 37(5)
37(12) 36(12) 37 (12) 36 (12) 37(13) 36(12)
38 (10) 38 (10) 35 (10) 38 (10) 37 (10) 37 (10)
37 (10) 36 (12) 38 (12) 36(11) 36(12) 37 (12)
37 (10) 37 (10) 38 (10) 35 (10) 37 (10) 37 (10)
312 314 342 329 324 302 292 322 280 294 314 298 291 230 256 241 282 232
114 125 111 114 126 117 112 90 109 99 117 93
271 271 252 255 250(11) 258
310* 287* 293* 246* 275* 295*
11/15/39 4 weeks
121 126 122 128 132 119
110 118 112 110 106 107
121 119 127 112 121 120
11/1/39 2 weeks
518 390 432 406 456 424
t
571 612 542 537 576
t
600* 612* 580* 592* 577*
535 529 488 463 390 493
498 (6) 549 (7) 461 (8) 501 325 (2)
11/29/39 6 weeks
868* 683* 746* 702* 780* 680*
848* 866* 826* 814* 839* 875*
815 680 (2) 808 (4) 918
1023 (3) 943 (8) 888 (10) 924(10) 1000 (1)
1212* 1090* 1121* 1092*
1132 (5) 1158(5)
846* 862* 813* 783 (10)* 722* 821* 902 (6) 879 908 (9) 847
1037 1066 1090 (2) 1024 (7) 680
12/27/39 10 weeks
845* 876* 784 (7)* 823* 530*
12/13/39 8 weeks
1300 (3)* 890* 1163 (3)* 1302*
1340* 1243* 1174* 1107* 1500*
1508 1210(8) 1213(8) 1213 (8)
1504*
1/10/40 12 weeks
Number in ( ) indicates the number of chicks on a given ration; this number remained constant until the number in the ( ) changed. All weights in grams. * Age when inoculated. t Indicates chicks not weighed.
Treatment
Lot
D a t e . . . .10/18/39 Age.... start
TABLE 3.—Eject of sulphur, E. tenella infection, and reinoculalion upon mean weight of chicks
1487 1110 1243 1450
1527 1363 (7) 1398 (9) 1363 (9) 1800
1115(4)
1/24/40 14 weeks
o
o >n O o r> o
H O
< w
H X H
t-
X > o o >1
o
c (-! *e X c w > a
o
w w w
o
28
0 . E. GOFF
younger ones as only one of the 12-weekold control chicks died when inoculated with 880,000 sporulated oocysts. The cause of this increased refractiveness to a second invasion of E. tenetta is unknown, but in these trials it may have been due to incomplete regeneration of glandular and cellular structures in the ceca, as Mayhew (1937) found considerable damage still evident six weeks after hemorrhage. However, the four conceivable theories concerned with the nature of immunity to coccidia as listed by Becker (1937) should not be overlooked. These are: (1) production of humoral antibodies; (2) coccidia may destroy the cells and a new type of cell unfavorable to the growth of the parasite be produced; (3) development of a local immunity due to physiological change in the epithelial cells; and (4) perhaps to a principle allied to the bacteriophage. The mean weight of chicks and gains made by chicks receiving identical rations for the same period of time were quite similar when uninfluenced by E. tenella infection, as shown on Table 3. Chicks receiving the control ration or the charcoal ration without sulphur tended to grow at a slightly faster rate than did those fed rations containing flowers of sulphur. A highly significant value of " F " was secured between the mean gains per chicks when the various rations were fed from the second through the fourth week. The difference in gains during the four- to six-week period were non-significant when the chicks were fed the basal diet for the first four weeks prior to receiving the various sulphurcontaining rations. Thus it appears that chicks four weeks of age have greater resistance to the toxic properties of flowers of sulphur than younger chicks. It is therefore indicated that up to 5 percent sulphur may be fed for a two-week period without materially influencing growth if the feeding of a sulphur-bearing ration is delayed until
the chicks are four weeks old. Subsequent sulphur irritations of the vent, the skin around the face, and under the wings were unimportant when this substance was included in the rations after the chicks were four weeks of age or older. The difference in the six- to eight-week gains between the inoculated and noninoculated chicks in groups C and D were highly significant. However, by referring to Table 3 it should be noted that the mean weight of group C chicks at six weeks of age was superior to the weight of Group D chicks in three instances. Thus their capacity for an increase in absolute gain was potentially greater. Therefore it is questionable just how much importance should be attached to this observation. This, however, is an indication that the sulphur and sulphur and charcoal rations gave added protection against the ravages of coccidiosis, and that desirable gains were secured in spite of E. tenella infection when sulphur was used. The differences in gains made between lots of all group D chicks for the 10- to 12-week period were not significant, which may be accounted for by the small numbers involved and the great variability between individuals within each lot. It should be noted that although the average weight per chick in this group was greatest for those fed 2 percent sulphur (602D) only 3 survived experimentally-produced coccidiosis, while 10 chicks remained in the lot receiving S percent sulphur (604D). Under farm conditions it is indicated that if chicks were grown until four weeks of age under conditions unfavorable to natural infection up to 5 percent flowers of sulphur might be fed after that time for at least two weeks without deleterious effect. If chicks are to be turned upon an infected range sulphur or sulphur and charcoal may be fed for a few days in advance so that protection from coccidiosis might be secured. Subse-
FLOWERS OF SULPHUR AND CHARCOAL IN THE PREVENTION OF COCCIDIOSIS
quent immunity due to infection and age may make it unnecessary to feed sulphur after the chicks are 12 to 16 weeks old. It is also indicated that in a natural outbreak of the disease S percent sulphur or 5 percent sulphur plus 5 percent charcoal might be fed at the time the first symptoms were observed in order to afford protection to un-infected chicks. There is no indication that sulphur has any value as a cure of coccidiosis. Sulphur, coupled with sanitation, should make a poultryman's effort to prevent coccidiosis more efficient and effective. SUMMARY
1. The data presented support previous investigations that the value of a ration containing 5 percent flowers of sulphur in preventing coccidiosis is increased when supplemented with 5 percent No. 10 oakwood charcoal. 2. A ration containing 2 percent flowers of sulphur plus S percent No. 10 oakwood charcoal was essentially equal to a ration containing 5 percent flowers of sulphur in preventing coccidiosis. 3. An initial infection with E. tenella increased refractiveness (immunity) to a subsequent inoculation. When measured in terms of mortality, however, the disease was not prevented. 4. Up to 5 percent flowers of sulphur was fed to chicks four weeks of age or older for a two-week period without deleterious effects. 5. Inclusion of charcoal in a basal ration
29
in the absence of flowers of sulphur was of no benefit in coccidiosis prevention. 6. It was indicated that chicks 12 weeks of age had greater resistance to coccidiosis than had chicks that were 18 or 28 days old. LITERATURE
CITED
Almquist, H. J., and D. Zander, 1940. Adsorbing charcoals in chick diets. Proc. Soc. Exp. Biol, and Med. 45:303-305. Becker, E. R., 1937. Coccidia and coccidiosis of domesticated game and laboratory animals and of man. Collegiate Press Inc. Ames, Iowa. Pp. 147. Dickinson, E. M., and R. H. Scofield, 1939. The effect of sulphur against artificial infection with Eimeria acervulina and Eimeria tenella. Poult. Sci. 18:419-431. Goff, 0 . E., and C. W. Upp, 1940. Effect of four grades of sulphur upon artificially produced coccidiosis (preliminary report). Poul. Sci. 19: 180-186. , 1940. The influence of elemental sulphur upon chick growth and bone ash. Poult. Sci. 19:270-280. Herrick, C. A., and C. E. Holmes, 1936. Effect of sulphur on coccidiosis in chickens. Vet. Med. 31:390-391. Holmes, C. E., J. H. Deobald, and C. A. Herrick, 1938. Sulphur and rickets. Poult. Sci. 17:136142. Mayhew, R. L., 1937. Studies on coccidiosis. IX. Histopathology of the caecal type in chickens. Trans, of Ann. Micro. Society 56:431-446. Snedecor, G. W., 1937. Statistical methods. Collegiate Press Inc. Ames, Iowa. Pp. 341. Van Es, L., and J. F. Olney, 1940. An inquiry into the influence of environment on the incidence of poultry diseases. Neb. Agr. Exp. Sta. Res. Bui. 118:26-40.
E.
GOFF
Louisiana State University, Agricultural Experiment Station, Baton Rouge, Louisiana (Received for publication May 12, 1941)
V
AN ES and Olney (1940) set forth the opinion of many poultrymen when they stated that, although coccidiosis might be controlled to a bearable extent by sanitation and management, success in its complete elimination by these measures was difficult. Herrick and Holmes (1936) and Dickinson and Scofield (1939) reported that flowers of sulphur aided in the prevention of Eimeria tenella infection. In a preliminary report Goff and Upp (1940a) found that the coccidiosis preventive properties of sulphur were enhanced when 5 percent charcoal was included in a ration containing 5 percent of any one of the four grades of sulphur studied. Some of the effects of sulphur upon the chicks have been reported by Holmes, Deobald, and Herrick (1938), Dickinson and Schofield (1939), and Goff and Upp (1940b). Additional data regarding the coccidiosis preventive properties of sulphur and charcoal have been collected and a part of these are presented in this paper. METHODS AND MATERIALS
The technic for preparing a suspension of coccidian oocysts for inoculation purposes as reported by Goff and Upp (1940a) has been simplified and consequently made more useable and rapid. Oocysts of Eimeria tenella were obtained by killing culture in* Published by approval of the director of the Louisiana Agricultural Experiment Station. This study was partially supported through a fellowship provided by the Freeport Sulphur Company.
fected chicks on the eighth or ninth day following inoculation. The ceca were removed, split, placed in Petri dishes, and moistened with a 2.5 percent solution of potassium dichromate. Cecal cores were crushed with a pair of forceps to aid aeration and sporulation. Few oocysts appeared to be present in the ceca at the end of the sixth day following inoculation; great numbers were present on the eighth day. Twelve days after inoculation many cecal cores had been passed and the number of oocysts present in the cecal content had greatly decreased; of those that were present the percent sporulation was usually low. Sporulation began by the eighteenth hour and continued through 48 hours. However, sporulation was practically complete after 24 hours, when the material was maintained at a temperature of approximately 75°F. After sporulation the best samples were combined, all ceca were scraped, and the material thus obtained ground through a 729-mesh per square inch wire sieve. Oocysts were concentrated from this material by repeated washing and straining and finally by sedimentation. Oocyst counts and inoculations of measured quantities of this inoculum were made in the manner described by Goff and Upp (1940a). This procedure has several advantages over the one previously presented in that it saves time, less expensive equipment is needed, oocysts are more numerous in cecal material
[23]
24
O. E. GOFF
than in droppings, oocysts have consistently sporulated better than those in cecal droppings, it is more pleasant to work with fresh cecal material, and the danger of contamination with other species of coccidia is greatly decreased. Flowers of sulphur and No. 10 hardwood oak charcoal were used in these trials. The charcoal is commonly referred to by the poultrymen as chick-size poultry charcoal. The adsorptive properties of this product are less than the finely divided product ordinarily used in biological laboratories for adsorption of vitamins, hormones, enzymes, and others, and appears to be a different product than the adsorbing charcoal used by Almquist and Zander (1940). All chicks were two weeks of age or older before they received any elemental sulphur. As indicated in Tables, 1, 2, and 3 the various lots were fed rations containing 2 or S percent flowers of sulphur with and without 5 percent No. 10 oakwood charcoal. One lot received S percent charcoal but no sulphur, while the control lots received the basal ration throughout with neither sulphur nor charcoal. All chicks were weighed individually every two weeks. All chicks were maintained in electrically heated battery brooders under conditions unfavorable to natural infection. The chicks used in June and July of 1939 were Single Comb White Leghorns and White Plymouth Rocks; those used in the later trials were White Plymouth Rocks. The basal ration used was the same as reported by Goff and Upp (1940a). Prior to the first inoculaton the cecal droppings of each chick were examined for oocysts, and in every case the findings were negative. At the time of hemorrhage the chicks were confined in individual chick batteries. As it is not known how many coccidian oocysts chicks might ingest under conditions favoring natural infection, it was deemed desirable to inoculate with large numbers
of sporulated oocysts to more rigidly test the coccidiosis preventive properties of flowers of sulphur. Thus, from 400,000 to almost 2,000,000 sporulated oocysts were administered each chick as indicated in Tables 1 and 2. RESULTS AND DISCUSSION
Data presented in Table 1 revealed that the chicks receiving flowers of sulphur (with or without charcoal) prior to inoculation with measured doses of E. tenella suffered less mortality from artificially induced coccidiosis than did chicks fed rations containing no sulphur. Highly consistent results were secured in each of the six trials. Chisquare tests revealed that no significant differences existed in coccidiosis mortality between the inoculated chicks receiving the basal ration and those receiving the ration containing 5 percent charcoal. All data involving chi-square were corrected for continuity by Yates' formula as suggested by Snedecor (1937). The chicks inoculated in each group and fed 2 percent flowers of sulphur plus S percent charcoal gave a greater percent survival than chicks receiving 2 percent sulphur without charcoal. This difference remained highly significant after correcting for continuity. Rations containing 2 percent sulphur proved to be inferior, verified statistically, to all other sulphur-containing rations in preventing coccidiosis mortality. When 5 percent charcoal was added to a ration containing 2 percent sulphur the number of chicks that survived the disease was essentially the same as those fed a ration containing 5 percent sulphur; chi-square tests revealed no significant difference. The appearance of the inoculated individuals and the degree of hemorrhage were quite similar for those chicks receiving 2 percent flowers of sulphur plus 5 percent charcoal or 5 percent sulphur without charcoal.
5
5% charcoal, no sulphur
5 5 5
4 5 1 1
4 5
3 5
1
Surv.
5
5 5
5 5
5
Inoc.
0
4 4
2 3
0
Surv.
No. chicks
6 weeks 8 weeks 1,980,000
7/1/39
10
10 10
10 10
10
2 10
10 10
10 10
6 7 8 10
10
0
Surv. Inoc. 10 10 10 10 10 10
0 6 10 10 10 0
Surv. Inoc. 45 45 45 45 45 45
0 7 8 10 10 1
2
X
3 5
X 2
Inoc.
Same controls used for D and E
X
5% charcoal, no sulphur
Group B chicks not included in totals.
9 9
9 10
5%|flowers of sulphur 5% flowers of sulphur plus 5% charcoal. X
6 7
42
Surv.
6 10 10 9
Surv. Inoc.
Surv
Inoc.
Surv.
No. chicks
12/14/39 B 2 weeks1st inoc. 8 weeks 840,000
Surv. 4 18 24 21 29 3
Reinoc. 15 18 27 29 34 3
Total chicks
1
100.0
72.4 85.3
100.0
16.0
Percent survival
11.1
88.9 97.8
62.2 84.4
6.7
X indicates no chicks available for second inoculation.
7 10
3 5 X
6 7
Inoc.
No. chicks
12/14/39 A 2 weeks 4 weeks 8 weeks 840,000
X 2
Surv.
No. chicks
No. chicks
No. chicks Inoc.
1/13/40 E 3 weeks 4 weeks 12 weeks 880,000
1/13/40 D 3 weeks 4 weeks 12 weeks 880,000
12/28/39 C 2 weeks 4 weeks 10 weeks 840,000
2% flowers of sulphur 2% flowers of sulphur plus 5% charcoal
Control (no sulphur). Never previously infected.
Experimental rations previously fed
Date inoculated Group designation Time off experimental ration Time elapsed between inoculations. Age of chick at second inoculation. Sporulated oocysts per chick
5
40 44
28 38
3
Percent Surv. survival
Total chicks
Surv. Inoc.
No. chicks
No. chicks
No. chicks Inoc.
12/14/39 D 4 weeks 8 weeks 840,000
11/30/39 C 2 weeks 6 weeks 780,000
11/19/39 A 2 weeks 4 weeks 780,000
TABLE 2.—Influence of an initial infection of E. tenella upon subsequent immunity, and carryover effect of sulphur
5 5
5 % flowers of sulphur 5 % flowers of sulphur plus 5% charcoal
5 5
4 5
5
2
5 5 S
Inoc.
No. chicks
No. chicks Surv.
6 weeks 8 weeks 660,000
2 weeks 4 weeks 400,000
Inoc.
7/1/39
6/2/39
2% flowers of sulphur
Experimental ration
Date inoculated Group designation Time on experimental diet before inoculation Age of chicks at time of inoculation Sporulated oocysts per chick
TABLE 1.—Value of flowers of sulphur in combination with poultry charcoal in coccidiosis prevention
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26
0. E. GOFF
After the data were corrected for continuity, differences in survival were not significant for chicks fed rations containing either 2 percent flowers of sulphur plus 5 percent charcoal, 5 percent sulphur, or 5 percent sulphur plus 5 percent charcoal. However, the percent survival appears to favor the chicks fed a ration containing 5 percent sulphur plus 5 percent charcoal. The degree of hemorrhage and the ill-appearance of the infected chicks was considered less severe in the 5 percent sulphur and charcoal group. Hemorrhage was observed usually in the no-sulphur lots prior to its occurrence in lots receiving sulphur. The chicks in the 5 percent sulphur and charcoal lot were inactive for a shorter period of time as a result of the disease than were chicks in the other lots; when disturbed these chicks would begin eating while the others would not. Thus the 5 percent sulphur and charcoal ration appeared to be superior to the other rations in preventing coccidiosis. The above data and observations further substantiate the author's belief that mortality alone is an inadequate measure of the severity of coccidiosis but may be used as one of the measures. Four weeks following the first inoculation all survivors were again inoculated with measured amounts of sporulated coccidian oocysts. The results of the reinoculations are recorded in Table 2. At the time of the second inoculations all chicks had received the basal ration without sulphur or charcoal for at least two weeks. Although a high percentage of the chicks survived this second inoculation, complete immunity was not established as all chicks shed oocysts and suffered slight or heavy hemorrhage which appeared quite similar in the various lots. At the time group A chicks were reinoculated (December 14, 1939), a number of group B chicks that had received identical rations, but reared coccidiosis-free, were
inoculated with the same inoculum and number of sporulated oocysts as recorded in Table 2. All group B chicks and control chicks of group A died of acute coccidiosis, while a majority of the reinoculated ones survived. Thus sulphur failed to exert any coccidiosis preventive properties two weeks after it had been removed from chick ra^ tions. At the time group D chicks were inoculated (December 14, 1939), all group E chicks were inoculated for the first time, after receiving the various rations as given in Table 3 for the preceding 22 hours. All group E chicks were given the same amount of inoculum as group D chicks (Table 1). Five chicks per lot were used except in the lot receiving 2 percent sulphur in which only four chicks were available for inoculation purposes. All chicks that received no flowers of sulphur died from acute coccidiosis. Of the chicks receiving 2 percent sulphur, one survived, two survived when fed 2 percent sulphur plus 5 percent charcoal, four survived when protected with 5 percent sulphur, while all five survived when protected with a ration containing 5 percent sulphur plus S percent charcoal. Additional data have been collected regarding this phase of the problem which will be presented in another paper. Reinoculation of chicks in groups C, D, and E provides additional evidence that increased resistance, immunity, or refractiveness was produced by an initial infection with E. tenella to a subsequent reinoculation. However, age undoubtedly influenced survival in groups D and E. At the time chicks in groups D and E were reinoculated (January 13, 1940), 5 chicks, 18 days of age, and 10 chicks, 28 days old, were inoculated with 440,000 sporulated oocysts from the same inoculum. All of these younger chicks died. These data indicate that resistance to coccidiosis was greater in 12-week-old chicks than in the
Control ration throughout. No sulphur. On 2 % F. of S. @ 2 wks. of age; off at 6 wks. On 2% F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5% F. of S. @ 2 wks.; off at 6 wks. On 5% F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % char. @ 2 wks.; off @ 6 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 2 wks.; off @ 6 wks. On 2 % F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % F. of S. at 2 wks.; off @ 6 wks. On 5 % F. of S . + 5 % char. @ 2 wks.; off @ 6 wks. On 5 % char, at 2 wks.; off @ 6 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 4 wks.; off @ 8 wks. On 2 % F. of S . + 5 % char. @ 4 wks.; off @ 8 wks. On 5 % F. of S. @ 4 wks.; off @ 8 wks. On 5 % F. of S . + 5 % char. @ 4 wks.; off @ 8 wks. On 5% char. @ 4 wks.; off @ 8 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 4 wks.; off @ 9 wks. On 2 % F. of S . + 5 % char. @ 4 wks.; off @ 9 wks. On 5% F. of S. @ 4 wks.; off @ 9 wks. On 5 % F. of S . + 5 % char. @ 4 wks.; off @ 9 wks. On 5% char. @ 4 wks.; off @ 9 wks.
Control ration throughout. No sulphur. On 2 % F. of S. @ 8 wks.; off @ 9 wks. On 2% F. of S . + 5 % char. @ 8 wks.; off @ 9 wks. On 5 % F. of S. @ 8 wks.; off @ 9 wks. On 5% F. of S . + 5 % char. @ 8 wks.; off @ 9 wks. On 5 % char. @ 8 wks.; off @ 9 wks.
601A 602A 603A 604A 605A 606A
601B 602B 603B 604B 605B 606B
601C 602C 603C 604C 605C 606C
601D 602D 603D 604D 605D 606D
601E 602E 603E 604E 605E 606E 35(5) 37(4) 38(5) 35(5) 36(5) 37(5)
37(12) 36(12) 37 (12) 36 (12) 37(13) 36(12)
38 (10) 38 (10) 35 (10) 38 (10) 37 (10) 37 (10)
37 (10) 36 (12) 38 (12) 36(11) 36(12) 37 (12)
37 (10) 37 (10) 38 (10) 35 (10) 37 (10) 37 (10)
312 314 342 329 324 302 292 322 280 294 314 298 291 230 256 241 282 232
114 125 111 114 126 117 112 90 109 99 117 93
271 271 252 255 250(11) 258
310* 287* 293* 246* 275* 295*
11/15/39 4 weeks
121 126 122 128 132 119
110 118 112 110 106 107
121 119 127 112 121 120
11/1/39 2 weeks
518 390 432 406 456 424
t
571 612 542 537 576
t
600* 612* 580* 592* 577*
535 529 488 463 390 493
498 (6) 549 (7) 461 (8) 501 325 (2)
11/29/39 6 weeks
868* 683* 746* 702* 780* 680*
848* 866* 826* 814* 839* 875*
815 680 (2) 808 (4) 918
1023 (3) 943 (8) 888 (10) 924(10) 1000 (1)
1212* 1090* 1121* 1092*
1132 (5) 1158(5)
846* 862* 813* 783 (10)* 722* 821* 902 (6) 879 908 (9) 847
1037 1066 1090 (2) 1024 (7) 680
12/27/39 10 weeks
845* 876* 784 (7)* 823* 530*
12/13/39 8 weeks
1300 (3)* 890* 1163 (3)* 1302*
1340* 1243* 1174* 1107* 1500*
1508 1210(8) 1213(8) 1213 (8)
1504*
1/10/40 12 weeks
Number in ( ) indicates the number of chicks on a given ration; this number remained constant until the number in the ( ) changed. All weights in grams. * Age when inoculated. t Indicates chicks not weighed.
Treatment
Lot
D a t e . . . .10/18/39 Age.... start
TABLE 3.—Eject of sulphur, E. tenella infection, and reinoculalion upon mean weight of chicks
1487 1110 1243 1450
1527 1363 (7) 1398 (9) 1363 (9) 1800
1115(4)
1/24/40 14 weeks
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28
0 . E. GOFF
younger ones as only one of the 12-weekold control chicks died when inoculated with 880,000 sporulated oocysts. The cause of this increased refractiveness to a second invasion of E. tenetta is unknown, but in these trials it may have been due to incomplete regeneration of glandular and cellular structures in the ceca, as Mayhew (1937) found considerable damage still evident six weeks after hemorrhage. However, the four conceivable theories concerned with the nature of immunity to coccidia as listed by Becker (1937) should not be overlooked. These are: (1) production of humoral antibodies; (2) coccidia may destroy the cells and a new type of cell unfavorable to the growth of the parasite be produced; (3) development of a local immunity due to physiological change in the epithelial cells; and (4) perhaps to a principle allied to the bacteriophage. The mean weight of chicks and gains made by chicks receiving identical rations for the same period of time were quite similar when uninfluenced by E. tenella infection, as shown on Table 3. Chicks receiving the control ration or the charcoal ration without sulphur tended to grow at a slightly faster rate than did those fed rations containing flowers of sulphur. A highly significant value of " F " was secured between the mean gains per chicks when the various rations were fed from the second through the fourth week. The difference in gains during the four- to six-week period were non-significant when the chicks were fed the basal diet for the first four weeks prior to receiving the various sulphurcontaining rations. Thus it appears that chicks four weeks of age have greater resistance to the toxic properties of flowers of sulphur than younger chicks. It is therefore indicated that up to 5 percent sulphur may be fed for a two-week period without materially influencing growth if the feeding of a sulphur-bearing ration is delayed until
the chicks are four weeks old. Subsequent sulphur irritations of the vent, the skin around the face, and under the wings were unimportant when this substance was included in the rations after the chicks were four weeks of age or older. The difference in the six- to eight-week gains between the inoculated and noninoculated chicks in groups C and D were highly significant. However, by referring to Table 3 it should be noted that the mean weight of group C chicks at six weeks of age was superior to the weight of Group D chicks in three instances. Thus their capacity for an increase in absolute gain was potentially greater. Therefore it is questionable just how much importance should be attached to this observation. This, however, is an indication that the sulphur and sulphur and charcoal rations gave added protection against the ravages of coccidiosis, and that desirable gains were secured in spite of E. tenella infection when sulphur was used. The differences in gains made between lots of all group D chicks for the 10- to 12-week period were not significant, which may be accounted for by the small numbers involved and the great variability between individuals within each lot. It should be noted that although the average weight per chick in this group was greatest for those fed 2 percent sulphur (602D) only 3 survived experimentally-produced coccidiosis, while 10 chicks remained in the lot receiving S percent sulphur (604D). Under farm conditions it is indicated that if chicks were grown until four weeks of age under conditions unfavorable to natural infection up to 5 percent flowers of sulphur might be fed after that time for at least two weeks without deleterious effect. If chicks are to be turned upon an infected range sulphur or sulphur and charcoal may be fed for a few days in advance so that protection from coccidiosis might be secured. Subse-
FLOWERS OF SULPHUR AND CHARCOAL IN THE PREVENTION OF COCCIDIOSIS
quent immunity due to infection and age may make it unnecessary to feed sulphur after the chicks are 12 to 16 weeks old. It is also indicated that in a natural outbreak of the disease S percent sulphur or 5 percent sulphur plus 5 percent charcoal might be fed at the time the first symptoms were observed in order to afford protection to un-infected chicks. There is no indication that sulphur has any value as a cure of coccidiosis. Sulphur, coupled with sanitation, should make a poultryman's effort to prevent coccidiosis more efficient and effective. SUMMARY
1. The data presented support previous investigations that the value of a ration containing 5 percent flowers of sulphur in preventing coccidiosis is increased when supplemented with 5 percent No. 10 oakwood charcoal. 2. A ration containing 2 percent flowers of sulphur plus S percent No. 10 oakwood charcoal was essentially equal to a ration containing 5 percent flowers of sulphur in preventing coccidiosis. 3. An initial infection with E. tenella increased refractiveness (immunity) to a subsequent inoculation. When measured in terms of mortality, however, the disease was not prevented. 4. Up to 5 percent flowers of sulphur was fed to chicks four weeks of age or older for a two-week period without deleterious effects. 5. Inclusion of charcoal in a basal ration
29
in the absence of flowers of sulphur was of no benefit in coccidiosis prevention. 6. It was indicated that chicks 12 weeks of age had greater resistance to coccidiosis than had chicks that were 18 or 28 days old. LITERATURE
CITED
Almquist, H. J., and D. Zander, 1940. Adsorbing charcoals in chick diets. Proc. Soc. Exp. Biol, and Med. 45:303-305. Becker, E. R., 1937. Coccidia and coccidiosis of domesticated game and laboratory animals and of man. Collegiate Press Inc. Ames, Iowa. Pp. 147. Dickinson, E. M., and R. H. Scofield, 1939. The effect of sulphur against artificial infection with Eimeria acervulina and Eimeria tenella. Poult. Sci. 18:419-431. Goff, 0 . E., and C. W. Upp, 1940. Effect of four grades of sulphur upon artificially produced coccidiosis (preliminary report). Poul. Sci. 19: 180-186. , 1940. The influence of elemental sulphur upon chick growth and bone ash. Poult. Sci. 19:270-280. Herrick, C. A., and C. E. Holmes, 1936. Effect of sulphur on coccidiosis in chickens. Vet. Med. 31:390-391. Holmes, C. E., J. H. Deobald, and C. A. Herrick, 1938. Sulphur and rickets. Poult. Sci. 17:136142. Mayhew, R. L., 1937. Studies on coccidiosis. IX. Histopathology of the caecal type in chickens. Trans, of Ann. Micro. Society 56:431-446. Snedecor, G. W., 1937. Statistical methods. Collegiate Press Inc. Ames, Iowa. Pp. 341. Van Es, L., and J. F. Olney, 1940. An inquiry into the influence of environment on the incidence of poultry diseases. Neb. Agr. Exp. Sta. Res. Bui. 118:26-40.