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The Effect of Sulphur Against Artificial Infection with Eimeria Acervulina and Eimeria Tenella
POULTRY S C I E N C E November, 1939, Vol. X V I I I , N o . 6
T h e Effect of Sulphur Against Artificial Infection with Eimeria Acervulina and Eimeria Tenella E. M. DICKINSON* Division of Veterinary Science, University of California R. H . SCOFIELD Los Angeles County Livestock Department Poultry Demonstration Plant, Pomona, California (Presented at annual meeting, Pullman, Washington, August, 1938; received for publication February 22, 1939)
H
ERRICK and Holmes (1936) have reported protective effects from feeding flowers of sulphur in a ration to chickens several days before they were artificially inoculated with a pure culture of Eimeria tenella, one of six species of coccidia that are known to infect chickens. These studies were undertaken to determine if sulphur would protect against inoculation with pure cultures of Eimeria acervulina as well as Eimeria tenella. E. acervulina was chosen because it is one of the species most frequently encountered infecting the small intestine, and has been shown by Johnson (1931) to adversely affect the health of chickens. In these investigations Single Comb White Leghorn chickens, of varying ages, were fed flowers of sulphur in a ration for six to eight days before and for six to eight days after they * Junior veterinarian, resigned April 30, 1938.
were inoculated with massive doses of sporulated occysts of either E. acervulina or E. tenella. The criteria used to estimate the effect on the birds were changes in body weight, feed consumption, clinical manifestations, mortality, and egg production. MATERIALS AND METHODS
Susceptible birds were provided by raising day-old chicks, SO to a brood, in clean electric brooders with floors of one-halfinch hardware cloth, and constructed so that there were no places for droppings to accumulate within reach of the chicks. Feed and water were reached on the outside of the brooder through an adjustable grating. At about five or six weeks of age the chicks were divided into lots of about 10 each and moved to clean metal cages 18 inches by 18 inches by 18 inches with a floor of rods (no. 6) spaced one inch
[419]
420
E. M.
DICKINSON AND R. H.
SCOFIELD
TABLE 1.—Response of cultures isolated to criteria for establishing species Species of Coccidia
Prepatent Predilection of Period Parasite
Lesions
Sporulation Time
Size in Microns
Shape Index
Eimeria acervulina
4 days 18 hours
Anterior and middle small intestine
Swollen intestinal mucosa. Whitish colonies of oocysts. Slimy mucoid feces.
18 to 20 hrs.
18.38X15.12* 0.82
Eimeria tenella
6 days 20 hours
Caeca and rectum
Sloughing of caecal mucosa with severe hemorrhage
24 to 36 hrs.
24.03X18.87* 0.78
* Average of 50 sporulated oocysts.
apart. Feed and water vessels were reached on the outside through a grating and at a height that droppings could not be passed into them. The brooding and rearing took place in a thoroughly cleaned isolation room constructed so as to keep out flies and other insects. All equipment, feed, and water used in the care of the susceptible birds were kept within the room. Periodic fecal examinations of the birds were uniformly negative for coccidial forms. The ration for all birds was mixed at a mill and delivered in new sacks to the rooms as needed. It consisted of the following: ground yellow corn, 15; ground wheat, 15; ground oats, 15; ground barley, 13; red bran, 10; fish meal (65 percent protein), 7.5; meat scrap (55 percent protein), 7.5; dry skimmilk, 5; alfalfa meal, 5; soybean meal, 3.75; limestone, 1.5; salt, 1; fortified vitamin A and D oil equivalent to 1 percent U.S.P. cod liver oil. To this ration flowers of sulphur was added in varying amounts. For inoculation purposes the birds were taken to another building and caged in one of two thoroughly cleaned rooms, one for E. acervulina and the other for E. tenella. These rooms were screened to keep out flies and other insects. Specially built individual batteries 19 inches by 11 inches by 15 inches with a floor of rods (No. 6) spaced 1% inches apart were used exclusively for the inoculated chickens, except for
one group of chicks that required the use of heated brooders (trial IV). Feed and water vessels were reached on the outside through a grating and at a height that droppings could not be passed into them. Each room had its own supply of equipment, feed, and water used in the care of the inoculated birds. The fact that all birds inoculated proved to be highly susceptible and uninoculated control birds kept in the same room likewise proved to be highly susceptible when later inoculated provided evidence that there was no accidental infection. The droppings were collected on paper which was changed daily. Cultures of E. acervulina and E. tenella used for inoculation were isolated from natural field cases and purified. They were considered as pure when they conformed to the criteria established by Tyzzer (1929) and Johnson (unpublished data), for these species as given in Table 1. To prepare cultures for inoculation, feces heavily laden with oocysts were mixed with enough 2.5 percent potassium dichromate to give the mixture the consistency of gruel. Thin layers of this mixture were poured into Petri dishes and incubated at room temperature for 7 to 10 days to allow the oocysts to sporulate. The mixture was then passed through fine sieves to remove coarse material and the oocysts were concentrated by sedimentation. All concentrated cultures were kept under refrigeration at about 40° F. The
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
inoculations of oocysts were made with graduated pipettes directly into the crop. The number of sporulated oocysts per cubic centimeter administered was estimated on the final culture by taking the average of from three to six direct microscopic counts of dilutions of the concentrated material. All inoculations with E. acervulina were made from cultures prepared on either March 22 or December 24, 1937. All inoculations with E. tenella were made from
421
the same culture which was prepared on August IS, 1937. RESULTS OF TRIALS WITH EIMERIA ACERVULINA
Three trials with E. acervulina were conducted with chickens 224, 144, and 78 days of age respectively for trials I, II, and III (Table 2). In trial I there were four lots of birds. Birds in lot 1 were inoculated individually with approximately 50,000,000 sporulated oocysts and were
TABLE 2.—Set-up of the trials with sulphur against Eimeria infections Dosage Num- Days of Inocu- Trial Lot Sex ber of of Oocysts lum Birds Age (Thousands) 1 2 3 4
50,000 50,000 50,000 50,000
9 9 9 9
3 3 4 3 7 5 7 5 7 5
224
0
224
35,000
Regular ration, no sulphur, inoculated controls.
3
9 d" 9 & 9
224
35,000
Regular ration, plus 10 percent sulphur 6 days before and 8 days after occcidial inoculation.
III
1 2 3
9 9 9
10 10 10
78 78 78
0 35,000 35,000
Regular ration, no sulphur, uninoculated controls. Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 6 days before and 8 days after coccidial inoculation.
IV
1 2 3
9 9 9
10 10 10
19 19 19
0 250 250
Regular ration, no sulphur, uninoculated controls Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
9
243
0
243
250
Regular ration, no sulphur, inoculated controls.
3
9 cf 9 c?
4 1 4 1 3 2
243
250
Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
1 2 3
9 9 9
8 8 10
133 133 133
0 250 250
Regular ration, no sulphur, uninoculated controls. Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
1
9 & 9 & ,9
7 5 7 5 6 6
279
0
279
250
Regular ration, no sulphur, inoculated controls.
279
250
Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
I
1 Eimeria acervulina
144 144 144 144
Treatment
II
2
1 V
2
Eimeria tenella VI
VII
2 3
&
Regular Regular Regular Regular
ration, no sulphur, inoculated controls. ration, plus 2 percent sulphur) fed 8 days before ration, plus 10 percent sulphur land 6 days after ration, plus 20 percent sulphur [coccidial inocula-
J tion. Regular ration, no sulphur, uninoculated controls.
Regular ration, no sulphur, uninoculated controls.
Regular ration, no sulphur, uninoculated controls.
422'
E. M. DICKINSON AND R. H. SCOFIELD TRIAL 1.
TRIAL 2. Lot 1(68.9 ot.l)
Lot 2(68.6 ci. 1 )
TRIAL S
# Sulphur discontinued in ratios » Inoculated with oocysts * Initial average body weight Days,-
"nr
10 ' ' ' " it ' '
"IF
'
'
*
•
"
"T—r—i
i
35
GRAPH 1. Average daily body weight of chickens in the trials with E. acervulina for 40 days after sulphur feeding was started.
fed only the regular ration. Lots 2, 3, and 4 were fed 2, 10, and 20 percent flowers of sulphur respectively in the regular ration for eight days before and six days after the birds were inoculated individually with approximately 50,000,000 sporulated oocysts. In trials II and III the set-
ups were the same with three lots of birds in each trial. Lot 1 served as uninoculated controls fed only the regular ration; lot 2 was fed only the regular ration and the birds were inoculated individually with approximately 35,000,000 sporulated oocysts; lot 3 was fed the regular ration
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
plus 10 percent flowers of sulphur six days before and eight days after the birds were inoculted individually with approximately 35,000,000 sporulated oocysts. All chickens inoculated in trials I, II, and III with E. acervulina, both those fed mash with 2, 10, 20 percent sulphur and the controls, developed severe infection.
423
The manifestations of the disease were: (1) decrease in food consumption and body weight (graphs 1 and 2), which started on the fourth day after inoculation, reached the lowest point on the ninth or tenth day, after which both were gradually regained and had returned to pre-inoculation level between the eighteenth and
TRIAL 1.
Lot 1(3.3 oi.*) Lot 2(2.0
oi.-)
Lot 3(3.0 oz.1)
Lot
§ 3 CO
TRIAL 2. Lot 1(2.7 oz.l)
TRIAL 3. Lot 1(2.4 oi.I)
Lot Lot
* Initial average feed consumption * Inoculated with oocysts
§ Sulphur discontinued in the ratio} Days
TF
ifc ' ' ' ' 2b
~W
"35"
~W
GRAPH 2. Average daily feed consumption of chickens in trials with E. acervulina for 40 days after sulphur feeding was started.
424
E. M. DICKINSON AND R. H. SCOFIELD
twenty-fourth days; (2) from the fourth to the seventh or eighth day after inoculation all inoculated birds became droopy, with ruffled feathers showing a typical clinical picture of chickens infected with coccidia. In addition, all inoculated birds voided copious amounts of slimy mucus with a meager amount of feces that was heavily laden with oocysts from the fifth to the ninth day after inoculation. After this, the feces returned to normal rather rapidly and there was a rapid reduction in the number of oocysts shed. (3) The egg production of the birds in trial I, the only ones which were laying, was completely stopped from the sixth to the twelfth day after inoculation after which it was gradually resumed. (4) Four birds of trial III died from E. acervulina infection. Two of these were inoculated controls (lot 2) and two were of the sulphur-fed group (lot 3). The deaths occurred on the sixth, twelfth, and fourteenth days after inoculation. No mortality from E. acervulina infection occurred among the chickens of trials I and II. The uninoculated controls (lots 1) of trials II and III remained normal in appearance and fecal examinations were consistently negative for coccidia. The mature birds of trial II maintained constant body weight and the growing chickens of trial III made regular gains in weight. Results of Reinoculation of Survivors in Trials II and III with E. acervulina. The birds which survived and recovered from the inoculation with E. acervulina in trials II and III were given a second inoculation of approximately 50,000,000 sporulated oocysts 100 days later. At this time the uninoculated controls (lot 1) of trials II and III were inoculated for the first time. During the interval all of these chickens had been kept in the cages and rooms as previously described and therefore had no
opportunity to pick up additional oocysts. The chickens inoculated consisted of 24 (14 females and 10 males) 334 days old and 20 females 178 days old which had been previously infected and 12 (7 females and 5 males) 334 days old and 10 females 178 days old which were infected for the first time. All chickens inoculated, whether for the first or second time, developed severe manifestations of the disease not unlike those described for the first inoculation. The loss in average body weight and feed consumption is shown in graph 3. Clinical manifestations were the same as those previously described. There was no mortality following this inoculation. In all lots egg production was either drastically reduced or completely stopped between the sixth and nineteenth day after inoculation. RESULTS OF TRIALS WITH EIMERIA TENELLA
Four trials with E. tenella were conducted with chickens 19, 243, 133, and 279 days of age respectively for trials IV, V, VI, and VII (Table 2). In these four trials the set-ups were the same with three lots of birds in each trial. Lot 1 consisted of the uninoculated control birds fed only the regular ration. The birds in lot 2 were fed only the regular ration and were individually inoculated with approximately 250,000 sporulated oocysts. The birds in lot 3 were fed the regular ration plus 10 percent flowers of sulphur seven days before and seven days after they were inoculated individually with approximately 2 50,000 sporulated oocysts. In trials IV, V, VI, and VII, all inoculated control birds (lot 2) developed severe manifestations of typical E. tenella infection which were: (1) Decrease in average body weight and feed consumption (graphs 4 and 5), which started on
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
425
TRIAL 2 " L o t 1(72.9
oi.*-)
* Initial average body or feed weight * Day of reinoculation with oocysts 25 30 20 35 IS 10 Davs 6 iv «2ter first inoculation with oocysts GRAPH 3. Average daily body weight and feed consumption of chickens in trials 2 and 3 following reinoculation with the same culture of E. acervulina.
the fourth or fifth day after inoculation and reached the lowest point between the seventh and eleventh day, after which both were gradually regained and had returned to preinoculation level between the tenth and twentieth day. (2) Between the fifth and the eighth day after inoculation, pro-
fuse hemorrhage was noted in the feces of all inoculated control birds. During this period the birds were droopy with the typical appearance of chickens infected with coccidia. Some birds did not pass normal caecal droppings for over two weeks after the hemorrhage had stopped,
426
E. M.
DICKINSON AND R.
H.
SCOFIELD
TRIAL 4
TRIAL 5
TRIAL 6
Lot
Lot
Lot 3(46.1 o » . i )
TRIAL 7
Lot 1(70.3 o s . » )
* Initial average body weight • Inoeulated with oocysts § Sulphur discontinued in ration Days
"T—
T T
16
To""
25
' 30
' '
55
GRAPH 4. Average daily body weight of chickens in the trials with E. tenella for 40 days after sulphur feeding was started.
427
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS TRIAL 4
Lot 1(1.8 oz.1)
TRIAL 6 Lot 1(4.4 os.*)
Lot 2(4.4 oz.*)
Lot 3(3.6 oz.*)
TRIAL 6
* Initial average feed consumption * Inooulatio* with oocysts § Sulphur discontinued in the ration
Days
' ' *ib
TT
'
'
*
•
SsT
35"
GRAPH 5. Average daily feed consumption of chickens in trials with E. tenella for 40 days after sulphur feeding was started.
although the small intestinal feces were normal and abundant in quantity. (3) Egg production of the inoculated control birds was drastically reduced in trials V and VII, the only trials in which the birds were laying. (4) In trials IV and VI, which included young birds, there was a mortality
of five and three birds respectively among the inoculated control birds (lot 2) due to E. tenella infection. In all trials, the sulphur-fed birds showed a marked protection against the E. tenella infection. In all trials, a few E. tenella oocysts were found in caecal
428
E. M. DICKINSON AND R. H. SCOFIELD
feces on the seventh day following inoculation. One 19-day-old chick in trial IV of the sulphur-fed lot 3 died from E. tenella infection on the sixth day after inoculation. This was the only fatality and the only gross evidence of E. tenella infection in the sulphur-fed birds. The uninoculated controls (lot 1) re-
a second time 41, 69, and 69 days, respectively, after the first inoculation. Birds that served as uninoculated controls (lot 1) during trials V, VI, and VII were inoculated for the first time. The same culture was used for all birds. Those of trial V received approximately 250,000 sporulated oocysts and those of trials VI and VII
glLot 1(58.6 oz.l)
1
Initial average body or feed weight * Day of reinooulation with oooysts
Days " ' 6 ' ' ID • ' 15 ' 20 ' ' 25 • • • ' i5U • ' ' S6 after f i r s t inoculation with oooysts GRAPH 6. Average daily body weight and feed consumption of chickens in trial S following reinoculation with the same culture of E. tenella.
mained normal in appearance, and fecal examinations were consistently negative for coccidia. Growing birds in trials IV and VI made regular gains, while mature birds in trials V and VII maintained constant body weight. Results of Reinoculation of Survivors in Trials V, VI, and VII with E. tenella. All birds in trials V, VI, and VII that survived the first inoculation were inoculated
approximately 500,000 sporulated oocysts. All chickens in trials V, VI, and VII were kept in the same room under conditions previously described during the interval between inoculations. No opportunity was allowed the birds to pick up additional oocysts. The chickens inoculated consisted of 15 females 202 days old, 24 (13 females, 11 males) 348 days old, and 10 (7 females, 3 males) 284 days old, which had
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
Days S 10 after f i r s t inoculation with oocysts
.15
20
26
30
429
35
GRAPH 7. Average daily body weight and feed consumption of chickens in trials 6 and 7 following reinoculation with the same culture of E. tenella.
been previously infected, and 8 females 202 days old, 12 (7 females, 5 males) 348 days old, and 5 (4 females, 1 male) 284
days old, which were infected for the first time. All chickens inoculated for the first time
430
E. M. DICKINSON AND R. H. SCOFIELD
(lot 1) and those that had previously been fed sulphur (lot 3) during the first inoculation developed very severe manifestations characteristic of E. tenella infection that have been described previously. The loss in daily body weight and feed consumption is presented in graphs 6 and 7. One bird from lot 1 that had served as an uninoculated control in trial V died from E. tenella infection seven days after inoculation. Two birds from lot 3 that had been fed sulphur in trial VI died from E. tenella infection seven days after inoculation. One bird from lot 1 that had served as an uninoculated control in trial VII died from E. tenella infection eight days after inoculation. Egg production from all females inoculated for the first time (lot 1) and those previously fed sulphur (lot 3) was drastically reduced between the seventh and twentieth day after inoculation, while egg production from all females that had served as inoculated controls (lot 2) during trials V, VI, and VII remained unaffected above 50 percent. * The chickens that had served as inoculated controls (lot 2) during trials V, VI, and VII in general showed no gross evidence of E. tenella. infection. However, in trial VII there were three birds which showed a trace of blood in the caecal droppings. These birds showed no drop in body weight; neither did it interfere with egg production. During these trials with E. tenella, it was observed that, although the prepatent period remained the same (oocysts appeared in the feces at the end of the seventh day), there was a noticeable difference in the time when blood was observed in the caecal feces. With a fresh culture about two or three weeks old, traces of hemorrhage were seen on the fourth day after inoculation which was followed by profuse hemorrhage on the
fifth, while the same culture approximately three months old or older when inoculated into susceptible chickens caused a trace of blood to show in the caecal feces on the fifth day that was followed by profuse hemorrhage on the sixth day after inoculation. It would appear from these observations that the age of the culture of E. tenella had an influence on the time when blood will appear in the feces. "SULPHUR RICKETS"
Holmes, Deobald, and Herrick (1938) report the occurrence of "sulphur rickets" when chicks are fed flowers of sulphur continuously for several weeks and are dependent solely upon cod liver oil for the antirachitic factor. In these trials flowers of sulphur was fed for only 14 days. Most of the birds were mature at the time they were placed on the feeding trials. However, the chicks used that were still growing showed no indication of rickets following this short period of sulphur feeding. Further, there was no perceptible difference in egg-shell texture noted in mature birds that were fed the flowers of sulphur for 14 days and those fed no flowers of sulphur. SUMMARY
In trials I, II, and III in which E. acervulina was used to infect chickens fed 2, 10, and 20 percent flowers of sulphur in the regular ration eight or six days before and six or eight days after inoculation with approximately SO or 35 million sporulated oocysts, a definite adverse effect was noted in all birds inoculated regardless of whether or not they had been fed flowers of sulphur in the ration. In trials I, II, and III in which Eacervidina was used to infect the birds, mortality as the result of the infection was noted only in trial III in which chicks 78 days of age were inoculated. There is an
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
indication from these trials that age may have some influence on the severity of the disease. In trials II and III, 100 days after the first inoculation of approximately 35,000,000 sporulated E. acervulina oocysts, a second inoculation of approximately 50,000,000 of the same culture was given to all surviving birds. The reaction, in so far as average body weight and feed consumption are concerned, indicates that no perceptible resistance or immunity had been developed from the first inoculation. In trials IV, V, VI, and VII in which E. tenella was used to infect chickens fed 10 percent flowers of sulphur in the regular ration seven days before and seven days after inoculation with approximately 250,000 sporulated oocysts, a definite protective influence was imparted to the birds fed the flowers of sulphur in this manner. All the other chickens inoculated at the same time with the same dosage and culture of E. tenella that were not fed flowers of sulphur developed severe manifestations of E. tenella infection. Birds protected by flowers of sulphur from E. tenella infection later proved to be highly susceptible to a dosage of approximately 250,000 or 500,000 sporulated E.
431
tenella oocysts, while previously inoculated control birds that developed severe manifestations of E. tenella infection proved to be highly resistant to the later inoculation. This would indicate that the protective effect imparted by flowers of sulphur took place before the sporozoites invaded the epithelial cells. Further, birds protected by sulphur shed only small numbers of oocysts in the feces. ACKNOWLEDGEMENT
We wish to acknowledge the splendid assistance given by Mr. Irving Denny, manager of the Los Angeles County Poultry Demonstration Plant, where this work was done, for his careful attention in handling the birds and the help he gave in designing and constructing the equipment. REFERENCES
Herrick, C. A., and C. E. Holmes, 1936. Effect of sulphur on coccidiosis in chickens. Vet. Med. 31:390-391. Johnson, W. T., 1931. Effect of five species of Eimeria upon egg production of Single Comb White Leghorns. Jour, of Parasitol. 18:122. Tyzzer, E. E., 1929. Coccidiosis of gallinaceous birds. Am. Jr. Hyg. 10:269-383. Holmes, C. E., J. H. Deobald, and C. A. Herrick, 1938. Sulphur and rickets, Poultry Sci. 17:136142
T h e Effect of Sulphur Against Artificial Infection with Eimeria Acervulina and Eimeria Tenella E. M. DICKINSON* Division of Veterinary Science, University of California R. H . SCOFIELD Los Angeles County Livestock Department Poultry Demonstration Plant, Pomona, California (Presented at annual meeting, Pullman, Washington, August, 1938; received for publication February 22, 1939)
H
ERRICK and Holmes (1936) have reported protective effects from feeding flowers of sulphur in a ration to chickens several days before they were artificially inoculated with a pure culture of Eimeria tenella, one of six species of coccidia that are known to infect chickens. These studies were undertaken to determine if sulphur would protect against inoculation with pure cultures of Eimeria acervulina as well as Eimeria tenella. E. acervulina was chosen because it is one of the species most frequently encountered infecting the small intestine, and has been shown by Johnson (1931) to adversely affect the health of chickens. In these investigations Single Comb White Leghorn chickens, of varying ages, were fed flowers of sulphur in a ration for six to eight days before and for six to eight days after they * Junior veterinarian, resigned April 30, 1938.
were inoculated with massive doses of sporulated occysts of either E. acervulina or E. tenella. The criteria used to estimate the effect on the birds were changes in body weight, feed consumption, clinical manifestations, mortality, and egg production. MATERIALS AND METHODS
Susceptible birds were provided by raising day-old chicks, SO to a brood, in clean electric brooders with floors of one-halfinch hardware cloth, and constructed so that there were no places for droppings to accumulate within reach of the chicks. Feed and water were reached on the outside of the brooder through an adjustable grating. At about five or six weeks of age the chicks were divided into lots of about 10 each and moved to clean metal cages 18 inches by 18 inches by 18 inches with a floor of rods (no. 6) spaced one inch
[419]
420
E. M.
DICKINSON AND R. H.
SCOFIELD
TABLE 1.—Response of cultures isolated to criteria for establishing species Species of Coccidia
Prepatent Predilection of Period Parasite
Lesions
Sporulation Time
Size in Microns
Shape Index
Eimeria acervulina
4 days 18 hours
Anterior and middle small intestine
Swollen intestinal mucosa. Whitish colonies of oocysts. Slimy mucoid feces.
18 to 20 hrs.
18.38X15.12* 0.82
Eimeria tenella
6 days 20 hours
Caeca and rectum
Sloughing of caecal mucosa with severe hemorrhage
24 to 36 hrs.
24.03X18.87* 0.78
* Average of 50 sporulated oocysts.
apart. Feed and water vessels were reached on the outside through a grating and at a height that droppings could not be passed into them. The brooding and rearing took place in a thoroughly cleaned isolation room constructed so as to keep out flies and other insects. All equipment, feed, and water used in the care of the susceptible birds were kept within the room. Periodic fecal examinations of the birds were uniformly negative for coccidial forms. The ration for all birds was mixed at a mill and delivered in new sacks to the rooms as needed. It consisted of the following: ground yellow corn, 15; ground wheat, 15; ground oats, 15; ground barley, 13; red bran, 10; fish meal (65 percent protein), 7.5; meat scrap (55 percent protein), 7.5; dry skimmilk, 5; alfalfa meal, 5; soybean meal, 3.75; limestone, 1.5; salt, 1; fortified vitamin A and D oil equivalent to 1 percent U.S.P. cod liver oil. To this ration flowers of sulphur was added in varying amounts. For inoculation purposes the birds were taken to another building and caged in one of two thoroughly cleaned rooms, one for E. acervulina and the other for E. tenella. These rooms were screened to keep out flies and other insects. Specially built individual batteries 19 inches by 11 inches by 15 inches with a floor of rods (No. 6) spaced 1% inches apart were used exclusively for the inoculated chickens, except for
one group of chicks that required the use of heated brooders (trial IV). Feed and water vessels were reached on the outside through a grating and at a height that droppings could not be passed into them. Each room had its own supply of equipment, feed, and water used in the care of the inoculated birds. The fact that all birds inoculated proved to be highly susceptible and uninoculated control birds kept in the same room likewise proved to be highly susceptible when later inoculated provided evidence that there was no accidental infection. The droppings were collected on paper which was changed daily. Cultures of E. acervulina and E. tenella used for inoculation were isolated from natural field cases and purified. They were considered as pure when they conformed to the criteria established by Tyzzer (1929) and Johnson (unpublished data), for these species as given in Table 1. To prepare cultures for inoculation, feces heavily laden with oocysts were mixed with enough 2.5 percent potassium dichromate to give the mixture the consistency of gruel. Thin layers of this mixture were poured into Petri dishes and incubated at room temperature for 7 to 10 days to allow the oocysts to sporulate. The mixture was then passed through fine sieves to remove coarse material and the oocysts were concentrated by sedimentation. All concentrated cultures were kept under refrigeration at about 40° F. The
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
inoculations of oocysts were made with graduated pipettes directly into the crop. The number of sporulated oocysts per cubic centimeter administered was estimated on the final culture by taking the average of from three to six direct microscopic counts of dilutions of the concentrated material. All inoculations with E. acervulina were made from cultures prepared on either March 22 or December 24, 1937. All inoculations with E. tenella were made from
421
the same culture which was prepared on August IS, 1937. RESULTS OF TRIALS WITH EIMERIA ACERVULINA
Three trials with E. acervulina were conducted with chickens 224, 144, and 78 days of age respectively for trials I, II, and III (Table 2). In trial I there were four lots of birds. Birds in lot 1 were inoculated individually with approximately 50,000,000 sporulated oocysts and were
TABLE 2.—Set-up of the trials with sulphur against Eimeria infections Dosage Num- Days of Inocu- Trial Lot Sex ber of of Oocysts lum Birds Age (Thousands) 1 2 3 4
50,000 50,000 50,000 50,000
9 9 9 9
3 3 4 3 7 5 7 5 7 5
224
0
224
35,000
Regular ration, no sulphur, inoculated controls.
3
9 d" 9 & 9
224
35,000
Regular ration, plus 10 percent sulphur 6 days before and 8 days after occcidial inoculation.
III
1 2 3
9 9 9
10 10 10
78 78 78
0 35,000 35,000
Regular ration, no sulphur, uninoculated controls. Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 6 days before and 8 days after coccidial inoculation.
IV
1 2 3
9 9 9
10 10 10
19 19 19
0 250 250
Regular ration, no sulphur, uninoculated controls Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
9
243
0
243
250
Regular ration, no sulphur, inoculated controls.
3
9 cf 9 c?
4 1 4 1 3 2
243
250
Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
1 2 3
9 9 9
8 8 10
133 133 133
0 250 250
Regular ration, no sulphur, uninoculated controls. Regular ration, no sulphur, inoculated controls. Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
1
9 & 9 & ,9
7 5 7 5 6 6
279
0
279
250
Regular ration, no sulphur, inoculated controls.
279
250
Regular ration, plus 10 percent sulphur 7 days before and 7 days after coccidial inoculation.
I
1 Eimeria acervulina
144 144 144 144
Treatment
II
2
1 V
2
Eimeria tenella VI
VII
2 3
&
Regular Regular Regular Regular
ration, no sulphur, inoculated controls. ration, plus 2 percent sulphur) fed 8 days before ration, plus 10 percent sulphur land 6 days after ration, plus 20 percent sulphur [coccidial inocula-
J tion. Regular ration, no sulphur, uninoculated controls.
Regular ration, no sulphur, uninoculated controls.
Regular ration, no sulphur, uninoculated controls.
422'
E. M. DICKINSON AND R. H. SCOFIELD TRIAL 1.
TRIAL 2. Lot 1(68.9 ot.l)
Lot 2(68.6 ci. 1 )
TRIAL S
# Sulphur discontinued in ratios » Inoculated with oocysts * Initial average body weight Days,-
"nr
10 ' ' ' " it ' '
"IF
'
'
*
•
"
"T—r—i
i
35
GRAPH 1. Average daily body weight of chickens in the trials with E. acervulina for 40 days after sulphur feeding was started.
fed only the regular ration. Lots 2, 3, and 4 were fed 2, 10, and 20 percent flowers of sulphur respectively in the regular ration for eight days before and six days after the birds were inoculated individually with approximately 50,000,000 sporulated oocysts. In trials II and III the set-
ups were the same with three lots of birds in each trial. Lot 1 served as uninoculated controls fed only the regular ration; lot 2 was fed only the regular ration and the birds were inoculated individually with approximately 35,000,000 sporulated oocysts; lot 3 was fed the regular ration
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
plus 10 percent flowers of sulphur six days before and eight days after the birds were inoculted individually with approximately 35,000,000 sporulated oocysts. All chickens inoculated in trials I, II, and III with E. acervulina, both those fed mash with 2, 10, 20 percent sulphur and the controls, developed severe infection.
423
The manifestations of the disease were: (1) decrease in food consumption and body weight (graphs 1 and 2), which started on the fourth day after inoculation, reached the lowest point on the ninth or tenth day, after which both were gradually regained and had returned to pre-inoculation level between the eighteenth and
TRIAL 1.
Lot 1(3.3 oi.*) Lot 2(2.0
oi.-)
Lot 3(3.0 oz.1)
Lot
§ 3 CO
TRIAL 2. Lot 1(2.7 oz.l)
TRIAL 3. Lot 1(2.4 oi.I)
Lot Lot
* Initial average feed consumption * Inoculated with oocysts
§ Sulphur discontinued in the ratio} Days
TF
ifc ' ' ' ' 2b
~W
"35"
~W
GRAPH 2. Average daily feed consumption of chickens in trials with E. acervulina for 40 days after sulphur feeding was started.
424
E. M. DICKINSON AND R. H. SCOFIELD
twenty-fourth days; (2) from the fourth to the seventh or eighth day after inoculation all inoculated birds became droopy, with ruffled feathers showing a typical clinical picture of chickens infected with coccidia. In addition, all inoculated birds voided copious amounts of slimy mucus with a meager amount of feces that was heavily laden with oocysts from the fifth to the ninth day after inoculation. After this, the feces returned to normal rather rapidly and there was a rapid reduction in the number of oocysts shed. (3) The egg production of the birds in trial I, the only ones which were laying, was completely stopped from the sixth to the twelfth day after inoculation after which it was gradually resumed. (4) Four birds of trial III died from E. acervulina infection. Two of these were inoculated controls (lot 2) and two were of the sulphur-fed group (lot 3). The deaths occurred on the sixth, twelfth, and fourteenth days after inoculation. No mortality from E. acervulina infection occurred among the chickens of trials I and II. The uninoculated controls (lots 1) of trials II and III remained normal in appearance and fecal examinations were consistently negative for coccidia. The mature birds of trial II maintained constant body weight and the growing chickens of trial III made regular gains in weight. Results of Reinoculation of Survivors in Trials II and III with E. acervulina. The birds which survived and recovered from the inoculation with E. acervulina in trials II and III were given a second inoculation of approximately 50,000,000 sporulated oocysts 100 days later. At this time the uninoculated controls (lot 1) of trials II and III were inoculated for the first time. During the interval all of these chickens had been kept in the cages and rooms as previously described and therefore had no
opportunity to pick up additional oocysts. The chickens inoculated consisted of 24 (14 females and 10 males) 334 days old and 20 females 178 days old which had been previously infected and 12 (7 females and 5 males) 334 days old and 10 females 178 days old which were infected for the first time. All chickens inoculated, whether for the first or second time, developed severe manifestations of the disease not unlike those described for the first inoculation. The loss in average body weight and feed consumption is shown in graph 3. Clinical manifestations were the same as those previously described. There was no mortality following this inoculation. In all lots egg production was either drastically reduced or completely stopped between the sixth and nineteenth day after inoculation. RESULTS OF TRIALS WITH EIMERIA TENELLA
Four trials with E. tenella were conducted with chickens 19, 243, 133, and 279 days of age respectively for trials IV, V, VI, and VII (Table 2). In these four trials the set-ups were the same with three lots of birds in each trial. Lot 1 consisted of the uninoculated control birds fed only the regular ration. The birds in lot 2 were fed only the regular ration and were individually inoculated with approximately 250,000 sporulated oocysts. The birds in lot 3 were fed the regular ration plus 10 percent flowers of sulphur seven days before and seven days after they were inoculated individually with approximately 2 50,000 sporulated oocysts. In trials IV, V, VI, and VII, all inoculated control birds (lot 2) developed severe manifestations of typical E. tenella infection which were: (1) Decrease in average body weight and feed consumption (graphs 4 and 5), which started on
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
425
TRIAL 2 " L o t 1(72.9
oi.*-)
* Initial average body or feed weight * Day of reinoculation with oocysts 25 30 20 35 IS 10 Davs 6 iv «2ter first inoculation with oocysts GRAPH 3. Average daily body weight and feed consumption of chickens in trials 2 and 3 following reinoculation with the same culture of E. acervulina.
the fourth or fifth day after inoculation and reached the lowest point between the seventh and eleventh day, after which both were gradually regained and had returned to preinoculation level between the tenth and twentieth day. (2) Between the fifth and the eighth day after inoculation, pro-
fuse hemorrhage was noted in the feces of all inoculated control birds. During this period the birds were droopy with the typical appearance of chickens infected with coccidia. Some birds did not pass normal caecal droppings for over two weeks after the hemorrhage had stopped,
426
E. M.
DICKINSON AND R.
H.
SCOFIELD
TRIAL 4
TRIAL 5
TRIAL 6
Lot
Lot
Lot 3(46.1 o » . i )
TRIAL 7
Lot 1(70.3 o s . » )
* Initial average body weight • Inoeulated with oocysts § Sulphur discontinued in ration Days
"T—
T T
16
To""
25
' 30
' '
55
GRAPH 4. Average daily body weight of chickens in the trials with E. tenella for 40 days after sulphur feeding was started.
427
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS TRIAL 4
Lot 1(1.8 oz.1)
TRIAL 6 Lot 1(4.4 os.*)
Lot 2(4.4 oz.*)
Lot 3(3.6 oz.*)
TRIAL 6
* Initial average feed consumption * Inooulatio* with oocysts § Sulphur discontinued in the ration
Days
' ' *ib
TT
'
'
*
•
SsT
35"
GRAPH 5. Average daily feed consumption of chickens in trials with E. tenella for 40 days after sulphur feeding was started.
although the small intestinal feces were normal and abundant in quantity. (3) Egg production of the inoculated control birds was drastically reduced in trials V and VII, the only trials in which the birds were laying. (4) In trials IV and VI, which included young birds, there was a mortality
of five and three birds respectively among the inoculated control birds (lot 2) due to E. tenella infection. In all trials, the sulphur-fed birds showed a marked protection against the E. tenella infection. In all trials, a few E. tenella oocysts were found in caecal
428
E. M. DICKINSON AND R. H. SCOFIELD
feces on the seventh day following inoculation. One 19-day-old chick in trial IV of the sulphur-fed lot 3 died from E. tenella infection on the sixth day after inoculation. This was the only fatality and the only gross evidence of E. tenella infection in the sulphur-fed birds. The uninoculated controls (lot 1) re-
a second time 41, 69, and 69 days, respectively, after the first inoculation. Birds that served as uninoculated controls (lot 1) during trials V, VI, and VII were inoculated for the first time. The same culture was used for all birds. Those of trial V received approximately 250,000 sporulated oocysts and those of trials VI and VII
glLot 1(58.6 oz.l)
1
Initial average body or feed weight * Day of reinooulation with oooysts
Days " ' 6 ' ' ID • ' 15 ' 20 ' ' 25 • • • ' i5U • ' ' S6 after f i r s t inoculation with oooysts GRAPH 6. Average daily body weight and feed consumption of chickens in trial S following reinoculation with the same culture of E. tenella.
mained normal in appearance, and fecal examinations were consistently negative for coccidia. Growing birds in trials IV and VI made regular gains, while mature birds in trials V and VII maintained constant body weight. Results of Reinoculation of Survivors in Trials V, VI, and VII with E. tenella. All birds in trials V, VI, and VII that survived the first inoculation were inoculated
approximately 500,000 sporulated oocysts. All chickens in trials V, VI, and VII were kept in the same room under conditions previously described during the interval between inoculations. No opportunity was allowed the birds to pick up additional oocysts. The chickens inoculated consisted of 15 females 202 days old, 24 (13 females, 11 males) 348 days old, and 10 (7 females, 3 males) 284 days old, which had
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
Days S 10 after f i r s t inoculation with oocysts
.15
20
26
30
429
35
GRAPH 7. Average daily body weight and feed consumption of chickens in trials 6 and 7 following reinoculation with the same culture of E. tenella.
been previously infected, and 8 females 202 days old, 12 (7 females, 5 males) 348 days old, and 5 (4 females, 1 male) 284
days old, which were infected for the first time. All chickens inoculated for the first time
430
E. M. DICKINSON AND R. H. SCOFIELD
(lot 1) and those that had previously been fed sulphur (lot 3) during the first inoculation developed very severe manifestations characteristic of E. tenella infection that have been described previously. The loss in daily body weight and feed consumption is presented in graphs 6 and 7. One bird from lot 1 that had served as an uninoculated control in trial V died from E. tenella infection seven days after inoculation. Two birds from lot 3 that had been fed sulphur in trial VI died from E. tenella infection seven days after inoculation. One bird from lot 1 that had served as an uninoculated control in trial VII died from E. tenella infection eight days after inoculation. Egg production from all females inoculated for the first time (lot 1) and those previously fed sulphur (lot 3) was drastically reduced between the seventh and twentieth day after inoculation, while egg production from all females that had served as inoculated controls (lot 2) during trials V, VI, and VII remained unaffected above 50 percent. * The chickens that had served as inoculated controls (lot 2) during trials V, VI, and VII in general showed no gross evidence of E. tenella. infection. However, in trial VII there were three birds which showed a trace of blood in the caecal droppings. These birds showed no drop in body weight; neither did it interfere with egg production. During these trials with E. tenella, it was observed that, although the prepatent period remained the same (oocysts appeared in the feces at the end of the seventh day), there was a noticeable difference in the time when blood was observed in the caecal feces. With a fresh culture about two or three weeks old, traces of hemorrhage were seen on the fourth day after inoculation which was followed by profuse hemorrhage on the
fifth, while the same culture approximately three months old or older when inoculated into susceptible chickens caused a trace of blood to show in the caecal feces on the fifth day that was followed by profuse hemorrhage on the sixth day after inoculation. It would appear from these observations that the age of the culture of E. tenella had an influence on the time when blood will appear in the feces. "SULPHUR RICKETS"
Holmes, Deobald, and Herrick (1938) report the occurrence of "sulphur rickets" when chicks are fed flowers of sulphur continuously for several weeks and are dependent solely upon cod liver oil for the antirachitic factor. In these trials flowers of sulphur was fed for only 14 days. Most of the birds were mature at the time they were placed on the feeding trials. However, the chicks used that were still growing showed no indication of rickets following this short period of sulphur feeding. Further, there was no perceptible difference in egg-shell texture noted in mature birds that were fed the flowers of sulphur for 14 days and those fed no flowers of sulphur. SUMMARY
In trials I, II, and III in which E. acervulina was used to infect chickens fed 2, 10, and 20 percent flowers of sulphur in the regular ration eight or six days before and six or eight days after inoculation with approximately SO or 35 million sporulated oocysts, a definite adverse effect was noted in all birds inoculated regardless of whether or not they had been fed flowers of sulphur in the ration. In trials I, II, and III in which Eacervidina was used to infect the birds, mortality as the result of the infection was noted only in trial III in which chicks 78 days of age were inoculated. There is an
EFFECT OF SULPHUR AGAINST ARTIFICIAL EIMERIA INFECTIONS
indication from these trials that age may have some influence on the severity of the disease. In trials II and III, 100 days after the first inoculation of approximately 35,000,000 sporulated E. acervulina oocysts, a second inoculation of approximately 50,000,000 of the same culture was given to all surviving birds. The reaction, in so far as average body weight and feed consumption are concerned, indicates that no perceptible resistance or immunity had been developed from the first inoculation. In trials IV, V, VI, and VII in which E. tenella was used to infect chickens fed 10 percent flowers of sulphur in the regular ration seven days before and seven days after inoculation with approximately 250,000 sporulated oocysts, a definite protective influence was imparted to the birds fed the flowers of sulphur in this manner. All the other chickens inoculated at the same time with the same dosage and culture of E. tenella that were not fed flowers of sulphur developed severe manifestations of E. tenella infection. Birds protected by flowers of sulphur from E. tenella infection later proved to be highly susceptible to a dosage of approximately 250,000 or 500,000 sporulated E.
431
tenella oocysts, while previously inoculated control birds that developed severe manifestations of E. tenella infection proved to be highly resistant to the later inoculation. This would indicate that the protective effect imparted by flowers of sulphur took place before the sporozoites invaded the epithelial cells. Further, birds protected by sulphur shed only small numbers of oocysts in the feces. ACKNOWLEDGEMENT
We wish to acknowledge the splendid assistance given by Mr. Irving Denny, manager of the Los Angeles County Poultry Demonstration Plant, where this work was done, for his careful attention in handling the birds and the help he gave in designing and constructing the equipment. REFERENCES
Herrick, C. A., and C. E. Holmes, 1936. Effect of sulphur on coccidiosis in chickens. Vet. Med. 31:390-391. Johnson, W. T., 1931. Effect of five species of Eimeria upon egg production of Single Comb White Leghorns. Jour, of Parasitol. 18:122. Tyzzer, E. E., 1929. Coccidiosis of gallinaceous birds. Am. Jr. Hyg. 10:269-383. Holmes, C. E., J. H. Deobald, and C. A. Herrick, 1938. Sulphur and rickets, Poultry Sci. 17:136142