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Studies on Fowl Typhoid
POULTRY SCIENCE November, 1949, Vol. XXVIII, No. 6 •
Studies on Fowl Typhoid II. Control of the Disease W. J. HALL, A. D. MACDONALD, AND D. H. LEGENHATJSEN Pathological Division, Bureau of Animal Industry, United States Department of Agriculture, BeltsviUe, Maryland (Received for publication February 11, 1949)
A
LTHOUGH fowl typhoid has been L recognized in this country for nearly half a century it was not until recent years that it became a major cause of poultry mortality. Control of the disease was relatively simple in the days when poultry raising was largely confined to small, widely scattered farm flocks. At that time control procedures consisted of culling sick birds, vaccination or depopulation, followed in some cases by cleaning and disinfection, and in others by merely depopulation for a variable period. Under those methods of poultry raising simple control methods were effective in keeping the disease in check. With the coming of mass poultry production such control procedures proved to be wholly inadequate. Losses in large breeding flocks and broiler plants mounted until the disease threatened the existence of the industry in some of the large poultry raising centers. I t became apparent that new methods of control must be developed that would be effective under conditions of mass production. Many foreign investigators including Panisset (1930), Coles (1946), Lesbouy-
ries (1941), Reis and Nobrega (1936), and Talavera (1946), advocate vaccination for the control of fowl typhoid. Kaupp and Dearstyne (1925), recommended the following procedures for the control of fowl typhoid: strict sanitation, destruction of sick birds, disinfection of drinking water, confinement of birds to new range, and vaccination of all healthy stock. Hammond (1945) was successful in controlling fowl typhoid in a breeding flock by the use of repeated whole blood rapid plate agglutination tests and the administration of sodium sulfathizaole in the drinking water. Preliminary studies in the field by means of the rapid whole blood agglutination test in 1944, and later by culture of all eggs laid by reactor (carrier) hens and exposure experiments, indicated to the authors (1949) that the key to the control of fowl typhoid in breeding flocks was the elimination of carriers together with the use of such sanitary procedures as seemed important under the circumstances. Other control measures such as immunization by vaccination and the administration of 789
790
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHATTSEN
bacteriophage, as well as chemotherapy, were also investigated. MANAGEMENT METHODS IN THE DELMARVA BROILER INDUSTRY
The following is a description of the management practices in the large poultry plant where the authors did most of their field work. Except for its unusually large size the operation of this plant with respect to management, housing and sanitation is typical of the industry in that area. 1. Husbandry.—This plant which has enjoyed a "puUorum clean" rating for several years, consists of 70,000 breeding birds, a hatchery of approximately 2,000,000 capacity, and a broiler plant with a capacity of over 2 million birds per year. In order to keep the hatchery running at capacity, hatching eggs are obtained from three separate sources, viz.: the home farm breeding plant, local flock owners, and 4 large breeding farms in other states. Eggs from all these sources are hatched in the large hatchery. An attempt is made to segregate the breeding flock replacement chicks in the hatchery by incubating and hatching them in a separate room. Upon hatching, the baby chicks are removed immediately to long brooder houses or, in the case of the breeding flock replacement chicks, to colony brooder houses with a capacity of 300 to 400 chicks each. The colony brooder houses are built of wood and have dirt floors. Each house has a capacity of 400 chicks. It is 16 feet by 16 feet and is built of light framing which is covered with tar paper and some wire netting. Heat is supplied by a coal burning stove. Water is supplied by an automatic fountain. The floor is covered with sand, which in turn is covered with about 4 inches of sawdust. The chicks are permitted outside in good weather. They are
kept in these brooder houses until about 12 weeks old when they are removed to a growing range, and at this time they are vaccinated against laryngotracheitis and pox. On the range they are put in A-shaped shelters which are open at one or both ends depending on the weather. Roosts are built about a foot above the ground. These shelters have a capacity of 100 birds. They are portable and are usually spotted about 150 feet apart. Feed hoppers are arranged in rows between each 2 rows of houses. Water is hauled in tank trucks and put in 50 gallon barrels from which it flows directly into automatic water troughs. Each feeding area has a water barrel. Breeder pullets are maintained on these ranges for about 10 weeks or until egg production is under way. Range capacity varies from 5,000 to 25,000 birds but the smaller units are favored. Some attempt is made to rotate ranges so as to permit cropping the land when space is available. In the latter part of the season a large part of the range is overgrown with weeds. After laying has begun, or as soon as a house is available, the pullets are removed to laying houses with a capacity of 5,000 to 6,000 birds each. The laying houses, of which there are 14, are widely scattered. They are arranged in groups of 2 to 5 and the various groups are in some cases several miles apart. The typical laying house has 20 pens, each pen being about 30 feet square. The pens are built in a straight line with a 2 story feed house and dwelling in the center separating the 2 groups of 10 pens, making a total length of approximately 800 feet. The houses are of the shed type with partially open front. Dropping pits are located along the rear wall. The floor is sand covered and has a top layer of about 4 inches of sawdust. The litter is not changed except that wet caked spots near the open windows or water
CONTROL or FOWL TYPHOID
fountains are removed. A platform car suspended from a track runs the entire length of the house, and is used to carry feed, eggs, coal, dead birds, etc. The broiler chicks are also reared in long 20 pen houses similar to the laying houses. Each room has two coal burning brooders with a capacity of 500 chickens per brooder. This arrangement permits each bird to have a space of J to f square foot. These birds remain in the house withoot change of litter, except as noted, until ready for market—usually 12 to 14 weeks. In good weather they are permitted to run into the yard in front of the house, the yard being about 40 feet wide and extending the full length of the house. Doors between pens are usually left open and thus there is a good deal of travel from pen to pen and especially toward the feed room. The large breeding flocks just described had a history of having suffered from fowl typhoid since their establishment about 3 years before. 2. Disease Control.—When fowl typhoid broke out in a broiler house or laying house, the birds in the room or rooms in which it broke out as well as in the adjoining rooms on either side were usually disposed of. This extensive culling procedure was followed in order to try to stop the disease by disposing of all birds, in the immediate vicinity of the outbreak, that might have been exposed. In any even, whenever a room or house was emptied whether disease was present or not, it was cleaned and disinfected in the following manner. All litter was removed down to the sand floor, and the top layer of sand was also removed. All feeding and watering equipment as well as nests and broody coops was removed and thoroughly scrubbed with water. The interior of the room—the walls, ceiling, and roosts—was also thor-
791
oughly scrubbed and washed down with a high pressure orchard spray hose. The room was then disinfected by spraying floors, walls, ceiling, and equipment with 2 percent lye solution. A layer of 2 to 3 inches of fresh sand and 4 to 5 inches of clean sawdust was applied to the floor before the room was refilled. 3. Sanitary Conditions.—-No special precautions were taken to prevent spread of disease from house to house or range to range by service or supervisory personnel. The same feed and water trucks were used on several ranges. No disinfectant solutions were maintained for disinfection of personnel travelling from one unit to another. Rats were numerous in some of the houses, where they burrowed into the litter. An attempt was made to control rats by poisoning but this was only partially successful. Turkey buzzards were seen on range, where they sought out dead birds and also availed themselves of water supplied the chickens. It was found that control of the various factors concerned in the spread of disease in such a large establishment was very difficult, if not impossible. EXPERIMENTAL 1. CONTROL BY AGGLUTINATION TESTING AND REMOVAL or REACTORS
As a demonstration of the effectiveness of repeated agglutination tests in removing typhoid carriers from a flock, Laying House No. 1 was selected. This house was tested 4 times by the authors at 30 day intervals by the rapid whole blood agglutination method using Strain 18 Salmonella gallinarum antigen. On the first test the number of reactors removed amounted to 1.5 percent and the 3 subsequent tests cleaned up the flock. After the removal of fowl typhoid reactors in flock No. 1 by the use of gallinarum plate antigen had
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W. J. HALL, A. D. MACDONALD AND D. H. LEGENHAUSEN
been demonstrated by. the authors the remaining 11 breeding flocks were tested with gallinarum plate antigen by the owner and all reactors, however slight, were removed. In order to make repeated tests of a large number of birds in a short time it seemed expedient to use the rapid whole blood plate method. Although S. pullorum and S. gallinarum cross agglutinate, it was desirable to determine which antigen would be most efficient in detecting
tests, strain 18 antigen seemed to be slightly more sensitive than K antigen. Later, however, the titers were lower with antigen 18 than with K antigen. Comparative tube tests with both pullorum and typhoid antigens were also made from time to time. Table 1 is a summary of these tests in which the two antigens (pullorum and gallinarum) as well as the test methods (tube and plate), are evaluated by the percentage of agreement as well as by the number
TABLE 1.—Comparison between antigens (pullorum and gallinarum) and test methods (tube and plate) in agglutination tests of typhoid reactors
Agreement between: AntigensfPullorum andlTube—Average of 2 tests \gallinarum J Plate—Average of 14 tests Test methods/Tube andlPullorum antigen —Average of 2 tests \plate /Gallinarum antigen—Average of 2 tests Total number of positive tests
Number tested
Number in agreement
Percent of agreement
91 839
79 789
86.8 94.0
91 91
83 81
91.2 89.0
Number tested
Number positive
Tube tests: Pullorum antigen —Average of 2 tests Gallinarum antigen—Average of 2 tests
91 91
79 65
Plate tests: Pullorum antigen —Average of 29 tests Gallinarum antigen—Average of 29 tests
839 839
714 681
typhoid infected birds since this was the first time that a gallinarum stained antigen had been used in the rapid whole blood test for the control of fowl typhoid. In order to make up as good a gallinarum antigen as possible, 21 strains of S. gallinarum obtained from the flocks mentioned above were screened for antigenicity. Strain 18 seemed to be one of the best antigenic strains, and an antigen was made from it. This and other gallinarum antigens were tested comparatively with S. pullorum K antigen on 30,000 birds in the field and at monthly intervals on the experimental typhoid reactor birds at the Bureau's laboratory at Beltsville, Maryland, over a period of 3 years. In the initial
Percent positive
.
86.8 71.4 85.1 81.1
of positive reactors detected. In this table the tube tests results are based on an average of only 2 tests whereas the plate test involved an average of 14 and 29 tests. This difference may account in part for the closer agreement between antigens in the plate tests (94.0 percent) than in the tube tests (86.8 percent). The agreement between test methods (tube and plate) was 91.2 percent when pullorum antigen was used and 89.0 percent when gallinarum antigen was used indicating that antigen employed had little effect on agreement between test methods in this instance. When the number of birds reacting positively was used as a criterion of effi-
CONTROL OF FOWL TYPHOID
ciency, in the tube tests pullorum antigen picked 86.8 percent of the total birds tested as reactors, and gallinarum antigen picked 71.4, a difference of 15.4 percent in favor of pullorum antigen. In the plate tests the difference in favor of pullorum antigen was only 4.0 percent. Here again the closer agreement between pullorum and gallinarum antigens in the number of positive reactors detected by the rapid plate method may be influenced by the large number of tests made. To sum up, pullorum antigen detected a slightly higher percentage of positive reactors than gallinarum in both the tube and plate tests. There was closer agreement between the plate tests than the tube tests when pullorum and gallinarum antigens were compared. As a result of a regional typhoid conference of research workers in poultry diseases, the States of Maryland, Delware, and Virginia agreed to furnish the Bureau with strains of Salmonella gallinarum to test for antigenicity. Of 72 strains sent in, 4 were selected as being most suitable, and these were used to make a polyvalent gallinarum antigen for use in the rapid whole blood plate test. This is known as Gallinarum Antigen No. 5. This antigen was tested comparatively with K (pullorum) antigen on the experimental typhoid reactor flocks at Beltsville for a number of months. It was also used at first to control typhoid in the large breeding flocks described above. Later, however, it was replaced by pullorum K antigen because the latter is a more highly refined antigen and it was more readily available. The flock owner tests each pullet flock as soon as it is put in the laying house, and again just before eggs are saved for hatching. DISCUSSION
In spite of the fact that each breeding flock is tested twice before eggs are saved
793
for hatching, each year there have been one or two typhoid "breaks" on the rearing ranges at about 16 to 20 weeks of age or at the time laying begins, but no breaks after housing. It seems probable that these breaks may be due to the consumption of infected eggs laid by pullets that were exposed to fowl typhoid in chickhood or infected directly through the eggs from their dams. Pullets begin to lay at 18 to 20 weeks of age. Eggs are dropped at various places on the range and some of them get broken. This fact may lead to egg eating and to a typhoid outbreak if infected eggs are consumed. On large ranges of 10,000 or more birds several hundred eggs a day may be found on the ground some of which are broken. That the broilers from these same flocks are not now subject to typhoid outbreaks is further confirmation of the egg origin of the breeding range outbreaks since broilers are sold before laying begins. However, the possibility that the disease was brought onto the range through some outside agency such as those mentioned in a previous paper on the subject (Hall, Legenhausen and MacDonald 1949) has not been ruled out. These range outbreaks are controlled by selling out the birds in the range shelters in the immediate vicinity of the outbreak or removing them to the laying house where the infected group may be segregated and sometimes treated with sulfonamides if losses continue. After recovery they are tested by the rapid plate method one or more times until all reactors are removed. By the use of these procedures losses have been kept to a very low figure so that fowl typhoid is no longer considered a major cause of death, whereas formerly thousands of birds, broilers as well as breeders, were lost annually. Needless to say, it is difficult to stamp out fowl typhoid in an establishment where 200,000 chicks or more are reared
794
W. J. H A L L , A. D . M A C D O N A L D AND D . H . L E G E N H A T J S E N
TABLE 2.—Results of vaccination against fowl typhoid Results of exposure Exper.
Date
Method of injection
Type of vaccine
Controls
Days vaccina, t o exp.
Method of exposure
No. exp./ no. dead FT
Percent
Aver. days exp. to death
5
2/11/44
Broth culture chlorof. killed
4 SC, 2 I D , 2 I P
11
2 drops S. gall, in conj.
8/6
75
9.3
12
3/31/44 3/31/44
Broth culture chlorof. killed
1, 2 & 5 cc. SC 1, 2 & 5 cc. per OS
12 12
1 cc. SC 1 cc. SC
3/0 3/2
67
26.0
Crystal violet killed
2 crop, 2 SC, 4 I M , 3 IP
58
0.5 cc. in crop 11/11 100
39
11/6/45
0.5 cc. in crop
3
3/3
7.4 8.3
42
1/29/46
Chloroform killed
Crop/4, I M / 4 , I P / 4
21
S. gall, in mash
12/8
67
9.3
43
1/22/46
Phenol killed
4 crop, 4 I M , 4 I P
28
S. gall, in mash
12/11
92
7.0
44
1/22/46
Formalin killed
4 crop, 4 I M , 4 I P
28
S. gall, in mash
12/12
100
6.5
45
1/24/46
Formalin killed in beeswax a n d peanut oil
4 IM, 4 I P
26
S. gall, in mash
8/8
100
7.0
46
1/24/46
Phenol killed in beeswax a n d peanut oil
4 I M , 4 I P , 6 SC
26
S. gall, in mash
14/13
93
7.3
49
2/5/46
Chloroform killed in beeswax a n d peanut oil
4 I M , 4 I P , 4 SC
14
S. gall, in mash
12/10
83
9.5
S. gall, in mash
12/9
75
7.2
12
Controls for Expers. 42, 43, 44, 45, 46, 49 57a
4/4/46
Brilliant green killed
1 I P , 2 I M , 1 cc.
19
S. gall. 1 cc. crop
3/1
33.3
30.6
57b
4/4/46
Brilliant green killed in beeswax a n d peanut oil
2 IP, 1 I M
19
S. gall. 1 cc. crop
5/3
60.0
30.6
• 58a
2/28/47
Merthiolate killed
IM l c c .
18
S. gall. 1 cc. 13/12 crop
58b
2/28/47
Merthiolate killed in beeswax a n d peanut oil
I M 1 cc:
18
S. gall. 1 cc. 15/15 crop
100
S. gall. & P . avicida heat-killed in 5 % phenol
1 cc. I M
29
40,000,000 S. gall. I M
15/14
93
9.4
40,000,000 S. gall. I M
5/3
60
7.0
40,000,000 S. gaU. I M
15/12
80
7.3
40,000,000 S. gall. I M
5/5
100
68a
12/2/46
Controls 68b
5 12/2/46
S. gall. & P . avicida heat-killed in 5 % phenol
18
lcc. IM
Controls
5 IM—Intramuscular IP—Intraperitoneal SC—Subcutaneous FT—Fowl typhoid ID—Intradermal
for breeding and to 2 to 3 million broilers are produced each year, and with a huge hatchery getting eggs from numerous flocks the fowl typhoid status of which is unknown. 2. CONTROL BY IMMUNIZATION
(a) Vaccination Immunization against fowl typhoid by vaccination has been practiced in this
92.3
8
P—Pasteurella Conj.—Conjunctival sac Exp.—Exposure S. gall.—Salmonella gallinarum
country and abroad for many years. Vaccination consisted in the injection of cultures of Salmonella gallinarum killed by various means such as heat, chemicals, etc. Immunization by the administration of bacteriophage has also been advocated, by D'Herelle (1926). Vaccination against fowl typhoid by the injection of cultures of S. gallinarum killed by heat or chemicals is generally
CONTROL OF FOWL TYPHOID
practiced in Europe, South Africa, and South America. Vaccination by the same method is also employed to a limited extent to control fowl typhoid in this country. However, there is a dearth of experimental evidence to substantiate its efficacy either in preventing or ameliorating an outbreak of the disease. Wilson (1946) found killed culture vaccines ineffective in protecting fowls against artificial exposure to S. gallinarum. This was true of either stock or autogenous vaccines. However, when a dose of autogenous vaccine was followed by a dose of live culture "a solid and lasting immunity" was produced. This last method of immunization was not recommended, however, owing to possibility of increase in virulence of live culture and consequent danger. .
EXPERIMENTAL
The authors have carried out 15 vaccination trials against fowl typhoid with results as shown in Table 2. In these trials, Salmonella gallinarum in 13 of these vaccines was killed by chemicals and in 2 it was killed by heat. In 5 of these trials the chemically killed vaccines were emulsified with an adjuvant, beeswax and peanut oil. This latter combination was used to try to prolong the absorption period and consequently the duration of immunity. Experiments 68a and 68b were carried out in collaboration with Dr. J. Talavera of Madrid, Spain, who made the vaccine. No controls were used in the first 3 experiments for the reason that these trials were largely exploratory. It was planned to repeat any experiments with adequate controls that showed evidence of protection in the preliminary trials. DISCUSSION
In general there was less total mortality among the vaccinated birds when the interval between vaccination and expo-
795
sure was three weeks or less than when the interval was more than three weeks. In experiments 5, 12, 42, 49, 57, 58, and 68b, where the interval between vaccination and exposure averaged 16.2 days, the average mortality of the vaccinated birds was 77.5 percent. In experiments 39, 43, 44, 45, 46, and 68a, where the interval between vaccination and exposure averaged 32.5 days, the average mortality of the vaccinated birds was 95.8 percent, whereas mortality of the controls on these experiments averaged 75 percent. The dose of vaccine administered in the different experiments varied from 0.5 cc. in the intradermal inoculation to 2 cc. by the parenteral routes. No difference could be noted in the results obtained whether the dose was .25 cc. or 2 cc. when administered by the parenteral routes. It may be argued that the method of exposure was too severe, but it is doubtful if administering the organism in the feed is much more severe than when hens eat infected eggs in the laying house. The TABLE 3.—Typhoid reactors out of a group of
55 that died of fowl typhoid Pen ^RirH No. 19A
« a a a
20A a a a a u a u a
16 67 6 17 ISIS
25 27 38 32 45 62 42 41 .43
Died
Titer
10-18-46 11-15-46 4- 1-47 1-25-47 1-31-48 2-27-47 3-28-47 3-19-47 11-29-46 10-23-46 11- 2-46 9- 8-46 5- 4-46 2- 6-46
4 3 3 3 4 4 3 4 4
1+
3+
3 3
Duration r> E g g of ° L „ _,•«.„_ transreaction (months) m i t t e r 12
7
14 15* 17 17 18 17 13 11 8* 10 5
4 4 Average 12.0
ingestion of infected eggs has already been shown in an earlier paper by the authors (1949) to cause very high mortality. From the above results it may be con-
796
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHAUSEN
eluded that any immunity resulting from vaccination is temporary, and that after a lapse of three or four weeks the bird may become susceptible again to the organism of fowl typhoid. That fowl typhoid immunity is of a temporary nature is further evidenced by the fact that of 55 birds reacting strongly to fowl typhoid for an average period of 12.0 months, 14 eventually died of acute fowl typhoid, as shown in Table 3. (b) Bacteriophagy D'Herelle (1926) cites numerous outbreaks of fowl typhoid in which he was successful in stopping losses by the injection of gallinarum bacteriophage. These were all farm outbreaks and no non-inoculated controls were kept. After a single injection of bacteriophage losses usually stopped abruptly or within a few days. In a few cases it was necessary to make a second inoculation to stop the outbreak. Kramer (1925) reported similar results in Holland. The experiments at Beltsville in bacteriophagy included two laboratory trials, (Experiments 38 and 60) and one field trial. The bacteriophage consisted of a combination of 3 strains which had been isolated from the feces of hens in 3 flocks in which outbreaks of fowl typhoid had occurred. These 3 strains, Tn, Ga, and Gr, had been serially passaged in broth 77, 51 and 62 times, respectively, before they were combined. Experiment 38 In this experiment 32 New Hampshire males 7 months old were given S. gallinarum cultures with or without bacteriophage by instillation into the crop. The S. gallinarum bacteriophage used was one that had previously been passaged 100 times in broth culture of S. gallinarum. The phage used in this experiment was prepared by adding 0.1 cc. of phage to 10
cc. of a 2\ hour broth culture of S. gallinarum and incubating at room temperature until the phage had removed all visible growth of S. gallinarum from the broth. These birds were divided into 3 groups as follows: Group 1 consisted of 8 birds which received 1 cc. broth culture of S. gallinarum 160T, cleared by phage plus 1 cc. of broth culture of S. gallinarum 1272T. Of the 8 birds treated all died of fowl typhoid in an average period of 8.1 days. Group 2 received the same treatment as Group 1 except that culture 1272T was used in both the phage-treated broth culture and in the control broth culture without phage. In this group 7 out of 8 died of fowl typhoid in an average period of 8.4 days. Group 3 (controls), consisting of 16 birds received 1 cc. broth culture 1272T, without phage. Of this group 14 out of 16 died of fowl typhoid in an average period of 7.3 days. From these experiments it is evident that in Groups 1 and 2 bacteriophage afforded no protection since the mortality was as great as in the control Group 3. 'Experiment 60 In this experiment the bacteriophage was added to the drinking water. Flasks containing 200 cc. of nutrient beef broth were inoculated with S. gallinarum (1360T) and incubated for 2 hours. One ml. of phage was then added to each flask and when the bacteria were lysed as indicated by clearing of the broth it was added to the drinking water at the rate of 1 flask to 2 quarts of water. In this experiment 31 birds were divided into 3 groups as follows: Group 1 consisted of 18 mature birds which received 200 cc. of broth culture of S. gallinarum 1360T with bacteriophage in 2 quarts of water. Group 2, consisting of 6 birds, received
797
CONTROL OF FOWL TYPHOID
the same amount of gallinarum culture in the drinking water but without phage. Since there were no deaths in either of these 2 groups after 34 days, they were both exposed to a virulent culture of S. gallinarum, 1590T, by feeding it in a moist mash. A control group of 7 birds was also exposed in the same manner with results as follows:
plate test, using gallinarum antigen 18, and removal of reactors. Two tests were made with the following results: Reactors removed Date
No. tested
Aug. 17 Sept. 20
2,600 2,261
No. 75 52
Percent 3.0 2.3
It was apparent in a month after the second test of this house that the use of No. treated/ the rapid whole blood test with gallinarum Days after No. died of Group No. exposure antigen and removal of reactors were not fowl typhoid effective in reducing the mortality in an 1 18/16 7.5 acute outbreak of the disease as it in2 6/4 7.0 3 (Controls) 7/6 12.8 creased after the last test until losses amounted to 30 to 40 birds a day. In Experiment 60 it is evident that culIt was then decided to try the effect of ture 1360T was avirulent since none of Group 2, which received no phage, died of gallinarum bacteriophage on mortality. fowl typhoid until after the challenge dose A bacteriophage which had been passaged of culture 1590T was administered 34 30 times in broth and was capable of cleardays later. In addition to being avirulent ing a broth culture in 3 to 4 hours was it appeared to be devoid of immunizing used. Although this phage had marked properties. Up to 34 days after phage initial bacteriostatic properties on gallitreatment no typhoid deaths had occurred narum cultures in vitro, it was unable to in either Groups 1 or 2, but within 7.5 prevent the resurgence of the more resistdays after feeding the challenge dose of ant organisms after 24 hours' incubation. 1590T a high percentage of birds in both It was, therefore, not a highly active groups were dead. S. gallinarum culture phage. However, it was used in the latter (1360T) had been carried on artificial part of October on pens 5, 6, and 7 on the media for 6 months prior to use in this west (typhoid tested) side, and in pen 14 experiment, whereas culture 1S90T had on the untested side of the house where been lyophilized immediately after bird mortality was lighter. In these 4 pens 582 birds were injected subcutaneously passage. with 1 cc. of bacteriophage. Daily morUse of bacteriophage in a field outbreak of tality records in the four pens for the following 6 days showed no diminution over fowl typhoid that in the control pens in the remainder Fowl typhoid broke out in a large 20 of the house. pen laying house (containing 5,000 to DISCUSSION 6,000 pullets) in a large breeding establishment in the latter part of August. Results from these experiments indiThe outbreak occurred on the west side cate that this Salmonella gallinarum bacof the house and involved about 10 pens, teriophage when injected or put in the or 2,600 pullets. drinking water was ineffective in protectI t was decided to use the west half of ing the birds exposed to the fowl typhoid this house first as a demonstration of the organism from fatal attacks of the diseffect on mortality in a flock outbreak of ease. Losses from the phage-treated and repeated agglutination tests by the rapid control groups were about the same.
798
W.
J. H A L L , A. D . M A C D O N A L D AND D . H . L E G E N H A U S E N
3. CONTROL BY CHEMOTHERAPY
I n recent years the sulfonamid drugs have been extensively tested for their curative value in a large number of diseases of animals including poultry. A few have proved to have distinct value in the
famerazine when fed in 1 percent amounts in the mash gave 88.9 percent protection to 18 birds, whereas only 25 percent of 20 controls survived. Holtman and Fisher (1947) succeeded in reducing mortality in chicks artifi-
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fflWff #ff T * : * l ffff # : # F i # # W -S : 10 11 12 0 1 2 3 4 5 6 ? 8 DAYS OF TREATMENT CHART 1. Effect of feeding sulfamerazine in mash on mortality due to fowl typhoid in broilers. (House S—20,000 chick capacity) control or cure of some diseases, while others have been of doubtful value. M a n y of them have a cumulative toxic effect and they should be used with caution. Moore (1948) reported on the use of sulfonamids in the control of fowl typhoid. Sulfonamid No. 2 gave 100 percent protection when fed in 0.2 percent amounts in the mash t o 12 birds. Sul-
cially infectedwith fowl typhoid by feeding sodium sulfamerazine, sodium sulfathiazole, or soluble sulfonamid No. 2 for 5 days. These drugs were most effective when administered in 0.2 to 0.3 percent amounts in the drinking water and immediately after infection. Sodium sulfamerazine given in 0.2 percent amounts in the drinking water for 5 days each month
CONTROL or FOWL TYPHOID
799
L'
CS24I
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 SEPTEMBER CHART 2. Effect of feeding sulfamerazine on mortality due to fowl typhoid in broilers (East half of House P—Capacity 10,000 birds)
for 2 months in a natural outbreak of fowl typhoid in 3 broiler plants reduced losses
from an average of 62 percent in the nontreated to 4 percent in the treated birds.
800
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHATJSEN
The same treatment when applied to mature reacting birds for 5 or 6 months tended to destroy the agglutination titer. Sulfatherapy-Field Trials.—While work ing on fowl typhoid in the field opportunity was had to test the effectiveness of some sulfonamid drugs on natural outbreaks of fowl typhoid in broiler flocks. Broiler House S was one of 20,000 chick capacity with 20 pens, 10 on the east side of the feed room and 10 on the west side. Losses from fowl typhoid were most severe on the west side so pens 1 to 10 were selected for experimental treatment with sulfonamids. The method of treatment and results are shown on Chart 1. The odd numbered pens, 1, 3, 5, 7 and 9 were treated while the even numbered pens were left as controls. In this sulfa feeding trial an attempt was made to determine the optimum dosage. In the D interval, pen 2, although in the control group, was treated because that pen was showing excessive mortality and the owner wished to save as many birds' as possible. The mortality curve for the treated group shows definite benefit from the treatment when compared with the curve for the non-treated group. However, when treatment was discontinued mortality in the treated group rose sharply. Mortality on these charts is total mortality and includes some deaths due to other diseases such as coccidiosis, but these were minor in comparison with those due to fowl typhoid. In House P, 20,000 capacity, mortality from fowl typhoid was heavy in pens 4 to 10 in the east half of this house prior to treatment (Chart 2). Pens 6 to 10 were given 1 percent sulfamerazine in the mash for 12 days while pens 1 to 5 were used as controls. Treatment was continued for 12 days near the end of which time there was a sudden sharp rise in mortality in the non-trveated group, pens 1 to 5. Mortality in the treated group, pens 6 to 10, remained comparatively low averaging 29.5
birds per day (0.6 percent) for 10 days, September 11 to 21, whereas mortality in the non-treated group averaged 331 birds per day (6.6 percent) for the same period. After 9 days without treatment there was a rise in mortality in the treated group. In these and other sulfonamid treatments of fowl typhoid in broilers and layers it is apparent that this drug suppresses mortality during and for a short time after the period of administration but that the organism survives the treatment and again multiplies after discontinuance of the drug. Hence, it appears that a method of intermittent treatment as suggested by Holtman and Fisher (1947) might be more successful. DISCUSSION
When earlier studies of the authors (1949) indicated that the typhoid reactor was the principal reservoir of the disease, it was evident that the disease could be controlled by destroying the reservoir by the elimination of carriers through the agglutination test or by depopulation. Other means of controlling losses such as vaccination and chemotherapy were considered as stopgap measures which, however, may be profitably employed in certain situations if found effective. These means of emergency control were, therefore, investigated. Chemotherapy may sometimes aid in controlling mortality due to fowl typhoid in broiler flocks until the birds are ready for market. However, sulfonamid drugs must be used with caution due to their cumulative toxic effect. Also lowered feed consumption where these drugs are used injudiciously resulting in weight losses must be guarded against. The sulfonamid drugs seem to be ineffective in some outbreaks for reasons which are not apparent. These drugs may be useful also in controlling mortality in laying flocks until the laying year is completed. Here again, however, the results may be disappointing in some outbreaks. Further, it should be emphasized that
801
CONTROL OF FOWL TYPHOID
such birds should not be retained as breeders as they may still be carriers even though they recover from an attack of the disease through the use of the drug. In view of the results of contaminated pen contact experiments reported by the authors in the first paper of this series (1949) it is doubtful if such elaborate cleaning and disinfecting procedures as are sometimes recommended are necessary since it appears that S. gallinarum loses virulence rather rapidly after removal from the animal body. However, until these theories are more definitely worked out reasonable sanitary precautions should be followed. SUMMARY
1. Complete control of fowl typhoid by repeated agglutination tests of breeders and elimination of reactors was accomplished in broiler flocks but not in breeder flocks reared on range. 2. The agglutination testing required to maintain a "pullorum clean" status was not sufficient to eliminate all fowl typhoid reactors. 3. Sanitary procedures could be carried out only to a limited extent in a large breeding and broiler establishment. 4. The relative importance of sanitary procedures in the control of fowl typhoid is not yet clear. 5. Comparative agglutination tests using both the plate and tube methods between pullorum and gallinarum antigens were made over a period of three years in flocks recovered from natural outbreaks and also in picked reactor flocks. These tests indicated that pullorum antigen consistently picked a slightly higher percentage of reactors than gallinarum antigen by both the tube and plate methods. 6. Results of 15 vaccination experiments using 13 chemically-killed and 2 heat-killed vaccines indicate that these prophylactic measures were of little value
in controlling mortality due to exposure to S. gallinarum. When the interval between vaccination and exposure was short (2-3 weeks) mortality was less than when the interval was over 3 weeks. After 30 days there was no indication of protection by vaccination. 7. The protective value of bacteriophage was tested in 2 experimental and 1 field trials. No beneficial results were noted as a result of the use of bacteriophage. 8. A number of field trials were made to test the effectiveness of the sulfonamid drugs in natural outbreaks of fowl typhoid in broilers. These indicated the sulfamerazine when fed in 0.5 to 1 percent concentration in the mash caused a marked drop in mortality due to fowl typhoid for about a week following treatment after which mortality gradually climbed again. REFERENCES Coles, J. D. W. A., 1946. Fowl typhoid. Farming in South Africa, 21:38-40. Hall, Legenhausen and MacDonald, 1949. Studies on Fowl Typhoid—I. Nature and Dissemination. Poultry Sci., 28: 344-362. Hammond, J. G., 1945. Sulfonamids in the control of Fowl Typhoid. Poultry Sci., 24:382-384. D'Herelle, F., 1926. The bacteriophage and its behavior. Baltimore, Williams & Wilkins. Holtman and Fisher, 1947. The application of sulfonamids to the control of typhoid in poultry. Poultry Sci., 26: 478-488. Kaupp, B. F., and Dearstyne, R. S., 1925. Fowl typhoid and fowl cholera. N. C. Agr. Exp. Sta. Tech. Bui. 27. Kramer, I. M., 1925. Quoted by D'Herelle. Lesbouyries, G., 1941. La Pathologie des Oiseaux. Vigot Freres, Paris, p. 387. Moore, E. N., 1948. The efficacy of recently developed sulfonamids against fowl typhoid. Poultry Sci., 25:307-311. Panisset, L., 1930. Diarrhie blanche bacillaire et typhose aviaire. 11th Internat. Vet. Cong., London. Reis, J., and P. Nobrega, 1936. Tratado de doensas das aves. Trabalho do Institute Biologico, Sao Paulo, Brazil. Talavera, J., 1946. Personal communications. Wilson, J. E., 1946. Fowl Typhoid. Certain aspects of the experimentally prodused disease. The Vet. Record, 58: 269-271.
Studies on Fowl Typhoid II. Control of the Disease W. J. HALL, A. D. MACDONALD, AND D. H. LEGENHATJSEN Pathological Division, Bureau of Animal Industry, United States Department of Agriculture, BeltsviUe, Maryland (Received for publication February 11, 1949)
A
LTHOUGH fowl typhoid has been L recognized in this country for nearly half a century it was not until recent years that it became a major cause of poultry mortality. Control of the disease was relatively simple in the days when poultry raising was largely confined to small, widely scattered farm flocks. At that time control procedures consisted of culling sick birds, vaccination or depopulation, followed in some cases by cleaning and disinfection, and in others by merely depopulation for a variable period. Under those methods of poultry raising simple control methods were effective in keeping the disease in check. With the coming of mass poultry production such control procedures proved to be wholly inadequate. Losses in large breeding flocks and broiler plants mounted until the disease threatened the existence of the industry in some of the large poultry raising centers. I t became apparent that new methods of control must be developed that would be effective under conditions of mass production. Many foreign investigators including Panisset (1930), Coles (1946), Lesbouy-
ries (1941), Reis and Nobrega (1936), and Talavera (1946), advocate vaccination for the control of fowl typhoid. Kaupp and Dearstyne (1925), recommended the following procedures for the control of fowl typhoid: strict sanitation, destruction of sick birds, disinfection of drinking water, confinement of birds to new range, and vaccination of all healthy stock. Hammond (1945) was successful in controlling fowl typhoid in a breeding flock by the use of repeated whole blood rapid plate agglutination tests and the administration of sodium sulfathizaole in the drinking water. Preliminary studies in the field by means of the rapid whole blood agglutination test in 1944, and later by culture of all eggs laid by reactor (carrier) hens and exposure experiments, indicated to the authors (1949) that the key to the control of fowl typhoid in breeding flocks was the elimination of carriers together with the use of such sanitary procedures as seemed important under the circumstances. Other control measures such as immunization by vaccination and the administration of 789
790
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHATTSEN
bacteriophage, as well as chemotherapy, were also investigated. MANAGEMENT METHODS IN THE DELMARVA BROILER INDUSTRY
The following is a description of the management practices in the large poultry plant where the authors did most of their field work. Except for its unusually large size the operation of this plant with respect to management, housing and sanitation is typical of the industry in that area. 1. Husbandry.—This plant which has enjoyed a "puUorum clean" rating for several years, consists of 70,000 breeding birds, a hatchery of approximately 2,000,000 capacity, and a broiler plant with a capacity of over 2 million birds per year. In order to keep the hatchery running at capacity, hatching eggs are obtained from three separate sources, viz.: the home farm breeding plant, local flock owners, and 4 large breeding farms in other states. Eggs from all these sources are hatched in the large hatchery. An attempt is made to segregate the breeding flock replacement chicks in the hatchery by incubating and hatching them in a separate room. Upon hatching, the baby chicks are removed immediately to long brooder houses or, in the case of the breeding flock replacement chicks, to colony brooder houses with a capacity of 300 to 400 chicks each. The colony brooder houses are built of wood and have dirt floors. Each house has a capacity of 400 chicks. It is 16 feet by 16 feet and is built of light framing which is covered with tar paper and some wire netting. Heat is supplied by a coal burning stove. Water is supplied by an automatic fountain. The floor is covered with sand, which in turn is covered with about 4 inches of sawdust. The chicks are permitted outside in good weather. They are
kept in these brooder houses until about 12 weeks old when they are removed to a growing range, and at this time they are vaccinated against laryngotracheitis and pox. On the range they are put in A-shaped shelters which are open at one or both ends depending on the weather. Roosts are built about a foot above the ground. These shelters have a capacity of 100 birds. They are portable and are usually spotted about 150 feet apart. Feed hoppers are arranged in rows between each 2 rows of houses. Water is hauled in tank trucks and put in 50 gallon barrels from which it flows directly into automatic water troughs. Each feeding area has a water barrel. Breeder pullets are maintained on these ranges for about 10 weeks or until egg production is under way. Range capacity varies from 5,000 to 25,000 birds but the smaller units are favored. Some attempt is made to rotate ranges so as to permit cropping the land when space is available. In the latter part of the season a large part of the range is overgrown with weeds. After laying has begun, or as soon as a house is available, the pullets are removed to laying houses with a capacity of 5,000 to 6,000 birds each. The laying houses, of which there are 14, are widely scattered. They are arranged in groups of 2 to 5 and the various groups are in some cases several miles apart. The typical laying house has 20 pens, each pen being about 30 feet square. The pens are built in a straight line with a 2 story feed house and dwelling in the center separating the 2 groups of 10 pens, making a total length of approximately 800 feet. The houses are of the shed type with partially open front. Dropping pits are located along the rear wall. The floor is sand covered and has a top layer of about 4 inches of sawdust. The litter is not changed except that wet caked spots near the open windows or water
CONTROL or FOWL TYPHOID
fountains are removed. A platform car suspended from a track runs the entire length of the house, and is used to carry feed, eggs, coal, dead birds, etc. The broiler chicks are also reared in long 20 pen houses similar to the laying houses. Each room has two coal burning brooders with a capacity of 500 chickens per brooder. This arrangement permits each bird to have a space of J to f square foot. These birds remain in the house withoot change of litter, except as noted, until ready for market—usually 12 to 14 weeks. In good weather they are permitted to run into the yard in front of the house, the yard being about 40 feet wide and extending the full length of the house. Doors between pens are usually left open and thus there is a good deal of travel from pen to pen and especially toward the feed room. The large breeding flocks just described had a history of having suffered from fowl typhoid since their establishment about 3 years before. 2. Disease Control.—When fowl typhoid broke out in a broiler house or laying house, the birds in the room or rooms in which it broke out as well as in the adjoining rooms on either side were usually disposed of. This extensive culling procedure was followed in order to try to stop the disease by disposing of all birds, in the immediate vicinity of the outbreak, that might have been exposed. In any even, whenever a room or house was emptied whether disease was present or not, it was cleaned and disinfected in the following manner. All litter was removed down to the sand floor, and the top layer of sand was also removed. All feeding and watering equipment as well as nests and broody coops was removed and thoroughly scrubbed with water. The interior of the room—the walls, ceiling, and roosts—was also thor-
791
oughly scrubbed and washed down with a high pressure orchard spray hose. The room was then disinfected by spraying floors, walls, ceiling, and equipment with 2 percent lye solution. A layer of 2 to 3 inches of fresh sand and 4 to 5 inches of clean sawdust was applied to the floor before the room was refilled. 3. Sanitary Conditions.—-No special precautions were taken to prevent spread of disease from house to house or range to range by service or supervisory personnel. The same feed and water trucks were used on several ranges. No disinfectant solutions were maintained for disinfection of personnel travelling from one unit to another. Rats were numerous in some of the houses, where they burrowed into the litter. An attempt was made to control rats by poisoning but this was only partially successful. Turkey buzzards were seen on range, where they sought out dead birds and also availed themselves of water supplied the chickens. It was found that control of the various factors concerned in the spread of disease in such a large establishment was very difficult, if not impossible. EXPERIMENTAL 1. CONTROL BY AGGLUTINATION TESTING AND REMOVAL or REACTORS
As a demonstration of the effectiveness of repeated agglutination tests in removing typhoid carriers from a flock, Laying House No. 1 was selected. This house was tested 4 times by the authors at 30 day intervals by the rapid whole blood agglutination method using Strain 18 Salmonella gallinarum antigen. On the first test the number of reactors removed amounted to 1.5 percent and the 3 subsequent tests cleaned up the flock. After the removal of fowl typhoid reactors in flock No. 1 by the use of gallinarum plate antigen had
792
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHAUSEN
been demonstrated by. the authors the remaining 11 breeding flocks were tested with gallinarum plate antigen by the owner and all reactors, however slight, were removed. In order to make repeated tests of a large number of birds in a short time it seemed expedient to use the rapid whole blood plate method. Although S. pullorum and S. gallinarum cross agglutinate, it was desirable to determine which antigen would be most efficient in detecting
tests, strain 18 antigen seemed to be slightly more sensitive than K antigen. Later, however, the titers were lower with antigen 18 than with K antigen. Comparative tube tests with both pullorum and typhoid antigens were also made from time to time. Table 1 is a summary of these tests in which the two antigens (pullorum and gallinarum) as well as the test methods (tube and plate), are evaluated by the percentage of agreement as well as by the number
TABLE 1.—Comparison between antigens (pullorum and gallinarum) and test methods (tube and plate) in agglutination tests of typhoid reactors
Agreement between: AntigensfPullorum andlTube—Average of 2 tests \gallinarum J Plate—Average of 14 tests Test methods/Tube andlPullorum antigen —Average of 2 tests \plate /Gallinarum antigen—Average of 2 tests Total number of positive tests
Number tested
Number in agreement
Percent of agreement
91 839
79 789
86.8 94.0
91 91
83 81
91.2 89.0
Number tested
Number positive
Tube tests: Pullorum antigen —Average of 2 tests Gallinarum antigen—Average of 2 tests
91 91
79 65
Plate tests: Pullorum antigen —Average of 29 tests Gallinarum antigen—Average of 29 tests
839 839
714 681
typhoid infected birds since this was the first time that a gallinarum stained antigen had been used in the rapid whole blood test for the control of fowl typhoid. In order to make up as good a gallinarum antigen as possible, 21 strains of S. gallinarum obtained from the flocks mentioned above were screened for antigenicity. Strain 18 seemed to be one of the best antigenic strains, and an antigen was made from it. This and other gallinarum antigens were tested comparatively with S. pullorum K antigen on 30,000 birds in the field and at monthly intervals on the experimental typhoid reactor birds at the Bureau's laboratory at Beltsville, Maryland, over a period of 3 years. In the initial
Percent positive
.
86.8 71.4 85.1 81.1
of positive reactors detected. In this table the tube tests results are based on an average of only 2 tests whereas the plate test involved an average of 14 and 29 tests. This difference may account in part for the closer agreement between antigens in the plate tests (94.0 percent) than in the tube tests (86.8 percent). The agreement between test methods (tube and plate) was 91.2 percent when pullorum antigen was used and 89.0 percent when gallinarum antigen was used indicating that antigen employed had little effect on agreement between test methods in this instance. When the number of birds reacting positively was used as a criterion of effi-
CONTROL OF FOWL TYPHOID
ciency, in the tube tests pullorum antigen picked 86.8 percent of the total birds tested as reactors, and gallinarum antigen picked 71.4, a difference of 15.4 percent in favor of pullorum antigen. In the plate tests the difference in favor of pullorum antigen was only 4.0 percent. Here again the closer agreement between pullorum and gallinarum antigens in the number of positive reactors detected by the rapid plate method may be influenced by the large number of tests made. To sum up, pullorum antigen detected a slightly higher percentage of positive reactors than gallinarum in both the tube and plate tests. There was closer agreement between the plate tests than the tube tests when pullorum and gallinarum antigens were compared. As a result of a regional typhoid conference of research workers in poultry diseases, the States of Maryland, Delware, and Virginia agreed to furnish the Bureau with strains of Salmonella gallinarum to test for antigenicity. Of 72 strains sent in, 4 were selected as being most suitable, and these were used to make a polyvalent gallinarum antigen for use in the rapid whole blood plate test. This is known as Gallinarum Antigen No. 5. This antigen was tested comparatively with K (pullorum) antigen on the experimental typhoid reactor flocks at Beltsville for a number of months. It was also used at first to control typhoid in the large breeding flocks described above. Later, however, it was replaced by pullorum K antigen because the latter is a more highly refined antigen and it was more readily available. The flock owner tests each pullet flock as soon as it is put in the laying house, and again just before eggs are saved for hatching. DISCUSSION
In spite of the fact that each breeding flock is tested twice before eggs are saved
793
for hatching, each year there have been one or two typhoid "breaks" on the rearing ranges at about 16 to 20 weeks of age or at the time laying begins, but no breaks after housing. It seems probable that these breaks may be due to the consumption of infected eggs laid by pullets that were exposed to fowl typhoid in chickhood or infected directly through the eggs from their dams. Pullets begin to lay at 18 to 20 weeks of age. Eggs are dropped at various places on the range and some of them get broken. This fact may lead to egg eating and to a typhoid outbreak if infected eggs are consumed. On large ranges of 10,000 or more birds several hundred eggs a day may be found on the ground some of which are broken. That the broilers from these same flocks are not now subject to typhoid outbreaks is further confirmation of the egg origin of the breeding range outbreaks since broilers are sold before laying begins. However, the possibility that the disease was brought onto the range through some outside agency such as those mentioned in a previous paper on the subject (Hall, Legenhausen and MacDonald 1949) has not been ruled out. These range outbreaks are controlled by selling out the birds in the range shelters in the immediate vicinity of the outbreak or removing them to the laying house where the infected group may be segregated and sometimes treated with sulfonamides if losses continue. After recovery they are tested by the rapid plate method one or more times until all reactors are removed. By the use of these procedures losses have been kept to a very low figure so that fowl typhoid is no longer considered a major cause of death, whereas formerly thousands of birds, broilers as well as breeders, were lost annually. Needless to say, it is difficult to stamp out fowl typhoid in an establishment where 200,000 chicks or more are reared
794
W. J. H A L L , A. D . M A C D O N A L D AND D . H . L E G E N H A T J S E N
TABLE 2.—Results of vaccination against fowl typhoid Results of exposure Exper.
Date
Method of injection
Type of vaccine
Controls
Days vaccina, t o exp.
Method of exposure
No. exp./ no. dead FT
Percent
Aver. days exp. to death
5
2/11/44
Broth culture chlorof. killed
4 SC, 2 I D , 2 I P
11
2 drops S. gall, in conj.
8/6
75
9.3
12
3/31/44 3/31/44
Broth culture chlorof. killed
1, 2 & 5 cc. SC 1, 2 & 5 cc. per OS
12 12
1 cc. SC 1 cc. SC
3/0 3/2
67
26.0
Crystal violet killed
2 crop, 2 SC, 4 I M , 3 IP
58
0.5 cc. in crop 11/11 100
39
11/6/45
0.5 cc. in crop
3
3/3
7.4 8.3
42
1/29/46
Chloroform killed
Crop/4, I M / 4 , I P / 4
21
S. gall, in mash
12/8
67
9.3
43
1/22/46
Phenol killed
4 crop, 4 I M , 4 I P
28
S. gall, in mash
12/11
92
7.0
44
1/22/46
Formalin killed
4 crop, 4 I M , 4 I P
28
S. gall, in mash
12/12
100
6.5
45
1/24/46
Formalin killed in beeswax a n d peanut oil
4 IM, 4 I P
26
S. gall, in mash
8/8
100
7.0
46
1/24/46
Phenol killed in beeswax a n d peanut oil
4 I M , 4 I P , 6 SC
26
S. gall, in mash
14/13
93
7.3
49
2/5/46
Chloroform killed in beeswax a n d peanut oil
4 I M , 4 I P , 4 SC
14
S. gall, in mash
12/10
83
9.5
S. gall, in mash
12/9
75
7.2
12
Controls for Expers. 42, 43, 44, 45, 46, 49 57a
4/4/46
Brilliant green killed
1 I P , 2 I M , 1 cc.
19
S. gall. 1 cc. crop
3/1
33.3
30.6
57b
4/4/46
Brilliant green killed in beeswax a n d peanut oil
2 IP, 1 I M
19
S. gall. 1 cc. crop
5/3
60.0
30.6
• 58a
2/28/47
Merthiolate killed
IM l c c .
18
S. gall. 1 cc. 13/12 crop
58b
2/28/47
Merthiolate killed in beeswax a n d peanut oil
I M 1 cc:
18
S. gall. 1 cc. 15/15 crop
100
S. gall. & P . avicida heat-killed in 5 % phenol
1 cc. I M
29
40,000,000 S. gall. I M
15/14
93
9.4
40,000,000 S. gall. I M
5/3
60
7.0
40,000,000 S. gaU. I M
15/12
80
7.3
40,000,000 S. gall. I M
5/5
100
68a
12/2/46
Controls 68b
5 12/2/46
S. gall. & P . avicida heat-killed in 5 % phenol
18
lcc. IM
Controls
5 IM—Intramuscular IP—Intraperitoneal SC—Subcutaneous FT—Fowl typhoid ID—Intradermal
for breeding and to 2 to 3 million broilers are produced each year, and with a huge hatchery getting eggs from numerous flocks the fowl typhoid status of which is unknown. 2. CONTROL BY IMMUNIZATION
(a) Vaccination Immunization against fowl typhoid by vaccination has been practiced in this
92.3
8
P—Pasteurella Conj.—Conjunctival sac Exp.—Exposure S. gall.—Salmonella gallinarum
country and abroad for many years. Vaccination consisted in the injection of cultures of Salmonella gallinarum killed by various means such as heat, chemicals, etc. Immunization by the administration of bacteriophage has also been advocated, by D'Herelle (1926). Vaccination against fowl typhoid by the injection of cultures of S. gallinarum killed by heat or chemicals is generally
CONTROL OF FOWL TYPHOID
practiced in Europe, South Africa, and South America. Vaccination by the same method is also employed to a limited extent to control fowl typhoid in this country. However, there is a dearth of experimental evidence to substantiate its efficacy either in preventing or ameliorating an outbreak of the disease. Wilson (1946) found killed culture vaccines ineffective in protecting fowls against artificial exposure to S. gallinarum. This was true of either stock or autogenous vaccines. However, when a dose of autogenous vaccine was followed by a dose of live culture "a solid and lasting immunity" was produced. This last method of immunization was not recommended, however, owing to possibility of increase in virulence of live culture and consequent danger. .
EXPERIMENTAL
The authors have carried out 15 vaccination trials against fowl typhoid with results as shown in Table 2. In these trials, Salmonella gallinarum in 13 of these vaccines was killed by chemicals and in 2 it was killed by heat. In 5 of these trials the chemically killed vaccines were emulsified with an adjuvant, beeswax and peanut oil. This latter combination was used to try to prolong the absorption period and consequently the duration of immunity. Experiments 68a and 68b were carried out in collaboration with Dr. J. Talavera of Madrid, Spain, who made the vaccine. No controls were used in the first 3 experiments for the reason that these trials were largely exploratory. It was planned to repeat any experiments with adequate controls that showed evidence of protection in the preliminary trials. DISCUSSION
In general there was less total mortality among the vaccinated birds when the interval between vaccination and expo-
795
sure was three weeks or less than when the interval was more than three weeks. In experiments 5, 12, 42, 49, 57, 58, and 68b, where the interval between vaccination and exposure averaged 16.2 days, the average mortality of the vaccinated birds was 77.5 percent. In experiments 39, 43, 44, 45, 46, and 68a, where the interval between vaccination and exposure averaged 32.5 days, the average mortality of the vaccinated birds was 95.8 percent, whereas mortality of the controls on these experiments averaged 75 percent. The dose of vaccine administered in the different experiments varied from 0.5 cc. in the intradermal inoculation to 2 cc. by the parenteral routes. No difference could be noted in the results obtained whether the dose was .25 cc. or 2 cc. when administered by the parenteral routes. It may be argued that the method of exposure was too severe, but it is doubtful if administering the organism in the feed is much more severe than when hens eat infected eggs in the laying house. The TABLE 3.—Typhoid reactors out of a group of
55 that died of fowl typhoid Pen ^RirH No. 19A
« a a a
20A a a a a u a u a
16 67 6 17 ISIS
25 27 38 32 45 62 42 41 .43
Died
Titer
10-18-46 11-15-46 4- 1-47 1-25-47 1-31-48 2-27-47 3-28-47 3-19-47 11-29-46 10-23-46 11- 2-46 9- 8-46 5- 4-46 2- 6-46
4 3 3 3 4 4 3 4 4
1+
3+
3 3
Duration r> E g g of ° L „ _,•«.„_ transreaction (months) m i t t e r 12
7
14 15* 17 17 18 17 13 11 8* 10 5
4 4 Average 12.0
ingestion of infected eggs has already been shown in an earlier paper by the authors (1949) to cause very high mortality. From the above results it may be con-
796
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHAUSEN
eluded that any immunity resulting from vaccination is temporary, and that after a lapse of three or four weeks the bird may become susceptible again to the organism of fowl typhoid. That fowl typhoid immunity is of a temporary nature is further evidenced by the fact that of 55 birds reacting strongly to fowl typhoid for an average period of 12.0 months, 14 eventually died of acute fowl typhoid, as shown in Table 3. (b) Bacteriophagy D'Herelle (1926) cites numerous outbreaks of fowl typhoid in which he was successful in stopping losses by the injection of gallinarum bacteriophage. These were all farm outbreaks and no non-inoculated controls were kept. After a single injection of bacteriophage losses usually stopped abruptly or within a few days. In a few cases it was necessary to make a second inoculation to stop the outbreak. Kramer (1925) reported similar results in Holland. The experiments at Beltsville in bacteriophagy included two laboratory trials, (Experiments 38 and 60) and one field trial. The bacteriophage consisted of a combination of 3 strains which had been isolated from the feces of hens in 3 flocks in which outbreaks of fowl typhoid had occurred. These 3 strains, Tn, Ga, and Gr, had been serially passaged in broth 77, 51 and 62 times, respectively, before they were combined. Experiment 38 In this experiment 32 New Hampshire males 7 months old were given S. gallinarum cultures with or without bacteriophage by instillation into the crop. The S. gallinarum bacteriophage used was one that had previously been passaged 100 times in broth culture of S. gallinarum. The phage used in this experiment was prepared by adding 0.1 cc. of phage to 10
cc. of a 2\ hour broth culture of S. gallinarum and incubating at room temperature until the phage had removed all visible growth of S. gallinarum from the broth. These birds were divided into 3 groups as follows: Group 1 consisted of 8 birds which received 1 cc. broth culture of S. gallinarum 160T, cleared by phage plus 1 cc. of broth culture of S. gallinarum 1272T. Of the 8 birds treated all died of fowl typhoid in an average period of 8.1 days. Group 2 received the same treatment as Group 1 except that culture 1272T was used in both the phage-treated broth culture and in the control broth culture without phage. In this group 7 out of 8 died of fowl typhoid in an average period of 8.4 days. Group 3 (controls), consisting of 16 birds received 1 cc. broth culture 1272T, without phage. Of this group 14 out of 16 died of fowl typhoid in an average period of 7.3 days. From these experiments it is evident that in Groups 1 and 2 bacteriophage afforded no protection since the mortality was as great as in the control Group 3. 'Experiment 60 In this experiment the bacteriophage was added to the drinking water. Flasks containing 200 cc. of nutrient beef broth were inoculated with S. gallinarum (1360T) and incubated for 2 hours. One ml. of phage was then added to each flask and when the bacteria were lysed as indicated by clearing of the broth it was added to the drinking water at the rate of 1 flask to 2 quarts of water. In this experiment 31 birds were divided into 3 groups as follows: Group 1 consisted of 18 mature birds which received 200 cc. of broth culture of S. gallinarum 1360T with bacteriophage in 2 quarts of water. Group 2, consisting of 6 birds, received
797
CONTROL OF FOWL TYPHOID
the same amount of gallinarum culture in the drinking water but without phage. Since there were no deaths in either of these 2 groups after 34 days, they were both exposed to a virulent culture of S. gallinarum, 1590T, by feeding it in a moist mash. A control group of 7 birds was also exposed in the same manner with results as follows:
plate test, using gallinarum antigen 18, and removal of reactors. Two tests were made with the following results: Reactors removed Date
No. tested
Aug. 17 Sept. 20
2,600 2,261
No. 75 52
Percent 3.0 2.3
It was apparent in a month after the second test of this house that the use of No. treated/ the rapid whole blood test with gallinarum Days after No. died of Group No. exposure antigen and removal of reactors were not fowl typhoid effective in reducing the mortality in an 1 18/16 7.5 acute outbreak of the disease as it in2 6/4 7.0 3 (Controls) 7/6 12.8 creased after the last test until losses amounted to 30 to 40 birds a day. In Experiment 60 it is evident that culIt was then decided to try the effect of ture 1360T was avirulent since none of Group 2, which received no phage, died of gallinarum bacteriophage on mortality. fowl typhoid until after the challenge dose A bacteriophage which had been passaged of culture 1590T was administered 34 30 times in broth and was capable of cleardays later. In addition to being avirulent ing a broth culture in 3 to 4 hours was it appeared to be devoid of immunizing used. Although this phage had marked properties. Up to 34 days after phage initial bacteriostatic properties on gallitreatment no typhoid deaths had occurred narum cultures in vitro, it was unable to in either Groups 1 or 2, but within 7.5 prevent the resurgence of the more resistdays after feeding the challenge dose of ant organisms after 24 hours' incubation. 1590T a high percentage of birds in both It was, therefore, not a highly active groups were dead. S. gallinarum culture phage. However, it was used in the latter (1360T) had been carried on artificial part of October on pens 5, 6, and 7 on the media for 6 months prior to use in this west (typhoid tested) side, and in pen 14 experiment, whereas culture 1S90T had on the untested side of the house where been lyophilized immediately after bird mortality was lighter. In these 4 pens 582 birds were injected subcutaneously passage. with 1 cc. of bacteriophage. Daily morUse of bacteriophage in a field outbreak of tality records in the four pens for the following 6 days showed no diminution over fowl typhoid that in the control pens in the remainder Fowl typhoid broke out in a large 20 of the house. pen laying house (containing 5,000 to DISCUSSION 6,000 pullets) in a large breeding establishment in the latter part of August. Results from these experiments indiThe outbreak occurred on the west side cate that this Salmonella gallinarum bacof the house and involved about 10 pens, teriophage when injected or put in the or 2,600 pullets. drinking water was ineffective in protectI t was decided to use the west half of ing the birds exposed to the fowl typhoid this house first as a demonstration of the organism from fatal attacks of the diseffect on mortality in a flock outbreak of ease. Losses from the phage-treated and repeated agglutination tests by the rapid control groups were about the same.
798
W.
J. H A L L , A. D . M A C D O N A L D AND D . H . L E G E N H A U S E N
3. CONTROL BY CHEMOTHERAPY
I n recent years the sulfonamid drugs have been extensively tested for their curative value in a large number of diseases of animals including poultry. A few have proved to have distinct value in the
famerazine when fed in 1 percent amounts in the mash gave 88.9 percent protection to 18 birds, whereas only 25 percent of 20 controls survived. Holtman and Fisher (1947) succeeded in reducing mortality in chicks artifi-
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fflWff #ff T * : * l ffff # : # F i # # W -S : 10 11 12 0 1 2 3 4 5 6 ? 8 DAYS OF TREATMENT CHART 1. Effect of feeding sulfamerazine in mash on mortality due to fowl typhoid in broilers. (House S—20,000 chick capacity) control or cure of some diseases, while others have been of doubtful value. M a n y of them have a cumulative toxic effect and they should be used with caution. Moore (1948) reported on the use of sulfonamids in the control of fowl typhoid. Sulfonamid No. 2 gave 100 percent protection when fed in 0.2 percent amounts in the mash t o 12 birds. Sul-
cially infectedwith fowl typhoid by feeding sodium sulfamerazine, sodium sulfathiazole, or soluble sulfonamid No. 2 for 5 days. These drugs were most effective when administered in 0.2 to 0.3 percent amounts in the drinking water and immediately after infection. Sodium sulfamerazine given in 0.2 percent amounts in the drinking water for 5 days each month
CONTROL or FOWL TYPHOID
799
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 SEPTEMBER CHART 2. Effect of feeding sulfamerazine on mortality due to fowl typhoid in broilers (East half of House P—Capacity 10,000 birds)
for 2 months in a natural outbreak of fowl typhoid in 3 broiler plants reduced losses
from an average of 62 percent in the nontreated to 4 percent in the treated birds.
800
W. J. HALL, A. D. MACDONALD AND D. H. LEGENHATJSEN
The same treatment when applied to mature reacting birds for 5 or 6 months tended to destroy the agglutination titer. Sulfatherapy-Field Trials.—While work ing on fowl typhoid in the field opportunity was had to test the effectiveness of some sulfonamid drugs on natural outbreaks of fowl typhoid in broiler flocks. Broiler House S was one of 20,000 chick capacity with 20 pens, 10 on the east side of the feed room and 10 on the west side. Losses from fowl typhoid were most severe on the west side so pens 1 to 10 were selected for experimental treatment with sulfonamids. The method of treatment and results are shown on Chart 1. The odd numbered pens, 1, 3, 5, 7 and 9 were treated while the even numbered pens were left as controls. In this sulfa feeding trial an attempt was made to determine the optimum dosage. In the D interval, pen 2, although in the control group, was treated because that pen was showing excessive mortality and the owner wished to save as many birds' as possible. The mortality curve for the treated group shows definite benefit from the treatment when compared with the curve for the non-treated group. However, when treatment was discontinued mortality in the treated group rose sharply. Mortality on these charts is total mortality and includes some deaths due to other diseases such as coccidiosis, but these were minor in comparison with those due to fowl typhoid. In House P, 20,000 capacity, mortality from fowl typhoid was heavy in pens 4 to 10 in the east half of this house prior to treatment (Chart 2). Pens 6 to 10 were given 1 percent sulfamerazine in the mash for 12 days while pens 1 to 5 were used as controls. Treatment was continued for 12 days near the end of which time there was a sudden sharp rise in mortality in the non-trveated group, pens 1 to 5. Mortality in the treated group, pens 6 to 10, remained comparatively low averaging 29.5
birds per day (0.6 percent) for 10 days, September 11 to 21, whereas mortality in the non-treated group averaged 331 birds per day (6.6 percent) for the same period. After 9 days without treatment there was a rise in mortality in the treated group. In these and other sulfonamid treatments of fowl typhoid in broilers and layers it is apparent that this drug suppresses mortality during and for a short time after the period of administration but that the organism survives the treatment and again multiplies after discontinuance of the drug. Hence, it appears that a method of intermittent treatment as suggested by Holtman and Fisher (1947) might be more successful. DISCUSSION
When earlier studies of the authors (1949) indicated that the typhoid reactor was the principal reservoir of the disease, it was evident that the disease could be controlled by destroying the reservoir by the elimination of carriers through the agglutination test or by depopulation. Other means of controlling losses such as vaccination and chemotherapy were considered as stopgap measures which, however, may be profitably employed in certain situations if found effective. These means of emergency control were, therefore, investigated. Chemotherapy may sometimes aid in controlling mortality due to fowl typhoid in broiler flocks until the birds are ready for market. However, sulfonamid drugs must be used with caution due to their cumulative toxic effect. Also lowered feed consumption where these drugs are used injudiciously resulting in weight losses must be guarded against. The sulfonamid drugs seem to be ineffective in some outbreaks for reasons which are not apparent. These drugs may be useful also in controlling mortality in laying flocks until the laying year is completed. Here again, however, the results may be disappointing in some outbreaks. Further, it should be emphasized that
801
CONTROL OF FOWL TYPHOID
such birds should not be retained as breeders as they may still be carriers even though they recover from an attack of the disease through the use of the drug. In view of the results of contaminated pen contact experiments reported by the authors in the first paper of this series (1949) it is doubtful if such elaborate cleaning and disinfecting procedures as are sometimes recommended are necessary since it appears that S. gallinarum loses virulence rather rapidly after removal from the animal body. However, until these theories are more definitely worked out reasonable sanitary precautions should be followed. SUMMARY
1. Complete control of fowl typhoid by repeated agglutination tests of breeders and elimination of reactors was accomplished in broiler flocks but not in breeder flocks reared on range. 2. The agglutination testing required to maintain a "pullorum clean" status was not sufficient to eliminate all fowl typhoid reactors. 3. Sanitary procedures could be carried out only to a limited extent in a large breeding and broiler establishment. 4. The relative importance of sanitary procedures in the control of fowl typhoid is not yet clear. 5. Comparative agglutination tests using both the plate and tube methods between pullorum and gallinarum antigens were made over a period of three years in flocks recovered from natural outbreaks and also in picked reactor flocks. These tests indicated that pullorum antigen consistently picked a slightly higher percentage of reactors than gallinarum antigen by both the tube and plate methods. 6. Results of 15 vaccination experiments using 13 chemically-killed and 2 heat-killed vaccines indicate that these prophylactic measures were of little value
in controlling mortality due to exposure to S. gallinarum. When the interval between vaccination and exposure was short (2-3 weeks) mortality was less than when the interval was over 3 weeks. After 30 days there was no indication of protection by vaccination. 7. The protective value of bacteriophage was tested in 2 experimental and 1 field trials. No beneficial results were noted as a result of the use of bacteriophage. 8. A number of field trials were made to test the effectiveness of the sulfonamid drugs in natural outbreaks of fowl typhoid in broilers. These indicated the sulfamerazine when fed in 0.5 to 1 percent concentration in the mash caused a marked drop in mortality due to fowl typhoid for about a week following treatment after which mortality gradually climbed again. REFERENCES Coles, J. D. W. A., 1946. Fowl typhoid. Farming in South Africa, 21:38-40. Hall, Legenhausen and MacDonald, 1949. Studies on Fowl Typhoid—I. Nature and Dissemination. Poultry Sci., 28: 344-362. Hammond, J. G., 1945. Sulfonamids in the control of Fowl Typhoid. Poultry Sci., 24:382-384. D'Herelle, F., 1926. The bacteriophage and its behavior. Baltimore, Williams & Wilkins. Holtman and Fisher, 1947. The application of sulfonamids to the control of typhoid in poultry. Poultry Sci., 26: 478-488. Kaupp, B. F., and Dearstyne, R. S., 1925. Fowl typhoid and fowl cholera. N. C. Agr. Exp. Sta. Tech. Bui. 27. Kramer, I. M., 1925. Quoted by D'Herelle. Lesbouyries, G., 1941. La Pathologie des Oiseaux. Vigot Freres, Paris, p. 387. Moore, E. N., 1948. The efficacy of recently developed sulfonamids against fowl typhoid. Poultry Sci., 25:307-311. Panisset, L., 1930. Diarrhie blanche bacillaire et typhose aviaire. 11th Internat. Vet. Cong., London. Reis, J., and P. Nobrega, 1936. Tratado de doensas das aves. Trabalho do Institute Biologico, Sao Paulo, Brazil. Talavera, J., 1946. Personal communications. Wilson, J. E., 1946. Fowl Typhoid. Certain aspects of the experimentally prodused disease. The Vet. Record, 58: 269-271.